Summer Vacation Studentship Reports 2015

Investigating Alzheimers disease in Down syndrome, using
transgenic mouse models

Aleksandra Lasica
Supervised by Frances K. Wiseman, Institute of Neurology, University College
London
Introduction: Individuals with Down

syndrome (DS), caused by trisomy of
chromosome 21 (Hsa21), have a
significantly elevated risk of developing
Alzheimers disease (AD). Virtually all
patients display the neuropathological
hallmarks of Alzheimers disease
amyloid plaques and neurofibrillary
tangles by the age of 30, and by the
age of 60 up to 70% of people with DS
develop dementia. This has been
attributed to triplication of the amyloid
precursor protein (APP) gene, present
on human chromosome 21. However,
recently it has been shown that genes on
Hsa21, other than APP, contribute to the
increased risk of AD in DS patients. The
Tc1 mouse model carries an extra copy of
Hsa21 but it is not functionally trisomic for
the APP gene. Cross between Tc1 model
and human mutant APP transgenic mouse
(J20) demonstrated that trisomy of
chromosome 21 is sufficient to exacerbate
APP/A pathology in the mouse brain
(Wiseman et al., unpublished data).
Currently, the main focus lies on the
identification of gene or genes contributing
to APP/A pathology.
assess whether the extra genes on

Hsa21, in the absence of APP, contribute
to AD pathology. During my internship I
was looking at the following phenotypic
features: number of amyloid plaques
(Ts2Yey) and APP levels (Ts3Yey) in
transgenic mouse models.
Methods:
Western blotting: Cortices from 3 monthold mice were homogenised in RIPA
buffer and total protein content of the
samples was determined by Bradford
assay. Subsequently, samples were
loaded onto NuPAGE Novex 4-12%
Bis-Tris Protein Gels) and SDS-PAGE gel
electrophoresis was performed at 150V for
75 mins in order to separate proteins.
Proteins were then transferred to
nitrocellulose membrane by electroblotting
at 35V for 2h at RT. Membranes were
then blocked in 5% milk in PBST
(phosphate-buffered saline (PBS) with
0.05% Tween-20 for 1 hour at RT, to
minimise non-specific binding. Primary
A8717 C-Terminal APP antibody (Sigma
Aldrich) (1:10,000) and primary A5441 actin antibody (Sigma Aldrich) (1:100,000)
were applied to the membrane overnight
at
4C.
Subsequently,
appropriate
horseradish peroxidase-linked secondary
antibodies were applied. Blots were
Aims: My project was aimed at

determining which regions on Hsa 21
potentially contribute to the APP/A
pathology, using transgenic mouse
models. In mouse genome, genes
orthologous to Hsa21 genes are split over
syntenic regions on chromosomes 16, 17
and 10. My project was focused on two
transgenic mouse models: Ts2Yeytrisomic for Hsa21 genes from mouse
chromosome 10 and Ts3Yey- trisomic for
Hsa21 genes from mouse chromosome
17. Both Ts2Yey and Ts3Yey were
crossed with J20 transgenic mouse. By
comparing the AD phenotype in J20 mice,
with
the
phenotype
observed
in
Ts2Yey/Ts3Yey*J20 mice, I was able to
developed using a film processor

(Xograph) and densitometric analysis of
bands was performed using ImageJ.
Plaque counting: Tissue was collected
from a cohort of mice at 6 months for
immunohistochemical assessment of 4G8positive A plaque load. Plaque counts
were recorded manually from both the
cortex and the hippocampus. The data
were analysed by repeat measures
ANOVA using SPSS software.
Figure 2: A plaque deposition in the Ts2Yey *J20 cross.

Hippocampus: tgAPP( F(1,27)=11.936, p=0.002), Ts2Yey
(F(1,21)=2.048, p=0.167) (with control groups excluded)
Cortex: tgAPP(F(1,27)=10.731,p=0.003)
Label tgAPP equivalent to J20 genotype.
Ts3Yey x J20 cross: APP levels
Results:
Ts2Yey x J20 cross:
A plaque
deposition
I was able to determine that in
accordance with previous data the
presence of the APP transgene (J20)
caused a significant increase in the
number of plaques in the cortex
(p=0.003) and hippocampus (p=0.002).
Ts2Yey trisomy did not significantly
influence the plaque load nor was an
interaction between Ts2Yey trisomy and
tgAPP observed. However, when WT
and Ts2Yey groups used as controls
were excluded from the analysis (based
on the absence of plaque deposition), a
slight trend (p=0.167) towards the
decrease in the number of plaques in the
hippocampus caused by Ts2Yey trisomy
was recorded. This possibly protective
role of Ts2Yey trisomy is further
supported by the data suggesting that
Ts2Yey trisomy rescues a tgAPPinduced sudden death phenotype and
causes an improvement in short- term
spatial
memory
(Pulford et
al.,
unpublished data).
Figure 3: Western blot

showing the levels of fulllength APP (FL-APP)
among all investigated
genotypes.
Signal intensity of the FL-APP band
was calculated relative to -actin
used as a loading control.
Statistical analysis of my Western blot

results indicated that as expected the
presence of tgAPP caused a
significant upregulation of APP
protein levels (p=0.00016). However,
no significant effect (p=0.521) of
Ts3Yey trisomy on the APP levels
was observed, which suggests that
trisomy of Hsa21 orthologous genes
on mouse chromosome 17, do not
increase APP protein levels in the
J20-tgAPP model system.
Figure 1: A deposition in hippocampus.

tgAPP
tgAPP*Ts2Yey
Figure 4: Levels of FL-APP relative to -actin.

tgAPP(F(1,20)=21.538, p=0.00016)
Ts3Yey(F(1,20)=0.427,p=0.521)
Acknowledgements
I would like to thank Professor
Elizabeth Fisher for accepting me into
the lab and Doctor Frances Wiseman
for providing me with the rounded
experience of research career. I
would also like to thank Karen
Cleverly and Laura Pulford for
teaching me the techniques that
enabled me to complete my project as
well as Matthew Rickman and Justin
Tosh for providing their technical
expertise.
Future directions:
Ts2Yey*J20 cross: In order to assess
possibly protective effect of Ts2Yey
trisomy on the A pathology, the plaque
deposition could be investigated at
different age time points, accompanied by
the increase in the sample size.
Ts3Yey*J20
cross:
In
order
to
investigate the mechanism of APP
pathology, the levels of C- terminal APP
fragments could be quantified using
Western blotting. This would indicate the
amount of APP processing that has
occurred. The attempt to quantify CTFs
was undertaken during my internship,
however due to time constraints and
problems with the primary antibody this
task was not completed.
The value of the studentship to the

student
This studentship was a truly enjoyable
experience that provided me with a
greater insight into the world of few
essential laboratory techniques as
well as boosting my confidence in
practical
skills.
However,
my
experience was not only limited to
data collection. I have learned the
basics of statistical analysis using
SPSS software, attended group
research meetings and presented my
data during one of the lab meetings.
The value of the studentship to the
laboratory
Aleks
has
made a
fantastic
contribution to our groups work this
summer, it was delightful to have her
enthusiasm in the lab and she worked
very hard. She worked particularly
closely with a second year PhD
student producing data of publishable
quality that we plan to submit for
publication next year. Aleks thus will
be a co-author on this paper, and we
would welcome her to work with us
again at any time.
Biochemical Society Studentship Report 2015
Structure-function relationship of the palmitoyl transferase DHHC5

Student: Aleksandra Tsolova; Supervisor: Dr Will Fuller, University of Dundee
Background
Massive endocytosis (MEND) is the major cause of injury during reperfusion of anoxic cardiac muscle. MEND occurs
following dynamic surface membrane protein palmitoylation (acylation) by palmitoyl transferase DHHC5, widely
recognized as one of very few cell-surface localized palmitoyl transferases. Calcium overload leading to mitochondrial
stress results in transient opening of the mitochondrial permeability transition pore (MPTP) and release of coenzyme-A
into the cytoplasm, where it is acylated to form a substrate for DHHC5. The enzyme then palmitoylates (reversibly
attaches a 16-carbon fatty acid to a cysteine thiol via a thioester bond) surface membrane proteins, causing them to
cluster together in lipid ordered domains, which leads to MEND, and thus, detrimental physiological effect on the cell. The
mechanisms underlying MEND are as-yet unidentified. Importantly, MEND occurs during reperfusion of anoxic cardiac
muscle, and is inhibited by interventions classically reported to reduce MPTP opening and protect against reperfusion
injury. DHHC5 knockout hearts, in which MEND is essentially absent, show significantly enhanced functional recovery
following anoxia-reperfusion, strongly implicating DHHC5 and the MEND pathway in reperfusion injury.
Interestingly, MEND is accelerated in the presence of the Na pump regulatory subunit phospholemman (PLM), which
1
inhibits the Na pump when palmitoylated , and activates the pump when phosphorylated. PLM is a substrate for DHHC5,
which is abundantly expressed in caveolar microdomains in cardiac muscle. Truncation analysis indicates interaction of
PLM with and palmitoylation of PLM by DHHC5 requires a region of the DHHC5 carboxyl terminus close to the
transmembrane domain (residues 218-334).
Figure 1: Mitochondrial release of CoA via MPTP during

reperfusion triggers massive internalisation of regions of cell
surface membrane, which leads to detrimental physiological
2
effect on the cell
Figure 2: DHHC5 structure showing DHHC domain, region of

substrate binding, and phosphorylation sites marked in red
(Source: Dr Will Fuller, University of Dundee, 2015)
Objectives
Investigate the structure-function relationship of DHHC5:
1. Narrow down the region of DHHC5 that interacts with its substrate PLM by performing additional truncations in the
region 218-334 of DHHC5.
2. Investigate the role of post-translational modifications in regulation of DHHC5 activity.
Description of the project
Key DHHC5 mutants were generated before the start of the project (C236A, C237A, C236/7A, N218X, C245X, S274X,
E304X)
1. Cell surface localization DHHC5 mutants
All DHHC5 point mutants were expressed in HEK cells, and cell surface proteins purified by streptavidin affinity
chromatography after application of membrane-impermeable biotinylation reagents.
2. Interaction of DHHC5 & PLM.
A long-term aim of the current research projects in the Fuller lab is to validate DHHC5 as a drug target for treatment of
ischemia-reperfusion injury in the heart. Identifying the precise region of DHHC5 that recognises individual substrates
may facilitate specific targeting of enzyme-substrate interactions. The HA tagged DHHC5 mutants were characterized
alongside wild type by investigating their association with PLM in transiently transfected HEK cells that stably express
3
PLM (described in ). DHHC5 was immunoprecipitated via the HA tag, and co-purifying PLM detected by western blotting.
2. Post-translational modifications of DHHC5.
The large carboxyl tail of DHHC5 includes multiple sites of both palmitoylation and phosphorylation, suggesting these
post-translational modifications may regulate DHHC5 activity. Therefore, the palmitoyl transferase activity of DHHC5
palmitoylation and phosphorylation point mutants was evaluated. DHHC5 is phosphorylated in mouse hearts at multiple
serine residues including S380, S432, and S636. Serine to alanine point mutants at these sites were generated for
characterization using the Agilent QuikChange site directed mutagenesis kit.
DHHC5 activity was assessed by expressing wild type and all mutant DHHC5 in a PLM-expressing DHHC5 knockout cell
line recently generated in Fuller laboratory. Palmitoylated proteins were purified by resin-assisted capture, and
palmitoylation of PLM assessed by western blotting.
Assessment of results and outcomes of the studentship

Various DHHC5 truncations were expressed in HEK cells stably transfected with PLM. All DHHC5 mutants were
successfully trafficked to the cell surface (Fig. 3). Hence no truncations interfered with the normal processing of DHHC5
in the secretory pathway. Despite small variations among results, after multiple repetitions of the experiments there was a
tendency of co-immunoprecipitation of the substrate PLM with DHHC5 mutants C245X, S274X, and E304X. This
suggests substrate-enzyme interaction at these sites (Fig. 4).
All serine to alanine point mutants S380, S432, and S636 were generated successfully and expressed in DHHC5 KO
cells. After purification of palmitoylated proteins by resin-assisted capture, it was found that palmitoylation of PLM varied
between constructs. The data revealed a close similarity in the level of palmitoylated protein among mutants (Fig. 5). This
was regarded as insufficient to provide a conclusion for the experiment. As a solution, a repeat should be performed of
the experiment to reveal a broader range of palmitoylated protein data. Due to time constrain a repetition set could not
have been made during current summer placement.
% PLM palmitoylation
Figure 3: Representative purification of cell

surface proteins by streptavidin affinity
chromatography. Cell surface proteins
were purified and immunoblotted as
indicated: (A) DHHC5 mutants expressed in
cells; (B) Cell surface fractions indicate all
DHHC5 mutants are localized on the cell
surface; (C) Localized cell surface Na-pump
confirming success of experiment
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Figure 4: Co-immunoprecipitation of
phospholemman and HA-tagged
DHHC5 mutants with anti-HA
sepharose beads. Constructs C245X,
S274X and E304X exhibit signal bands,
suggesting for association and
interaction of phospholemman at
these specific mutants.
Figure 5: Relative palmitoylation of phospholemman in DHHC5 KO

cells compared to wild-type (WT) normalized to 100%. The
increasing/decreasing expression is equivalent to the instance of
palmitoylation taking place at the specific expression site of the
mutant. Control sample was not transfected and thus did not
contain exogenous nucleic acids.
E304X
S274X
C245X
N218X
Control
S636A
S420A
S380A
C236/7A
C237A
C236A
WT
Future direction
The project can be taken for further characterization of
the substrate-binding region. As DHHC5 substrate, PLM,
is highly polar, a modification in amino acids in binding
region could be made. Positively charged amino acids
could be changed for negatively charged ones and
substrate-enzyme interaction assessed. Following the
same principle, negatively charged amino acids could be altered into positively charged and activity of the enzyme to bind
PLM assessed once again. Furthermore, as there are 6 phosphorylation sites identified, serine residues of these sites
can be amended into glutamate/aspartate residues which would initiate pseudo-phosphorylation.
Departures from original proposal
No major departures from the original proposal.
Contribution of grant to personal career aspirations and personal value of studentship
Throughout my summer placement in Fuller lab I not only gained and mastered major techniques while working in the lab
as well as I improved my research, writing and presenting skills, but I also understood the importance of organizing my
time to be efficient and scheduling my work to fit time constraints and still be productive. I learnt that sets of trial and error
underlie a successful experiment and in order to achieve good results I had to be patient and able to troubleshoot. Now, I
am much more confident in working in lab environment, analyzing results and communicating in a scientific language,
and I am eager to apply my knowledge again in the future. This invaluable experience will significantly aid me while I
progress further in my qualifications and build my career as a scientific research leader.
Value of the Studentship to the lab
Aleksandra did an excellent job and produced some high quality data while working in the Fuller lab. Her work
characterising DHHC5 truncations clearly showed that all the mutants she characterised trafficked correctly through the
secretory pathway. Unfortunately, our DHHC5 KO cell line grows too slowly for anyone to do any meaningful work with it,
but the experiments Aleksandra was able to perform showed good consistency with the co-immunoprecipitation data,
which has undoubtedly helped us to map the site of interaction of PLM & DHHC5. The phosphorylation site mutants that
Aleksandra produced are currently being characterised and will be a very valuable resource for this project as it moves
forward.
Related work and recommended literature
1.
2.
3.
Howie J, Tulloch LB, Shattock MJ, et al. Biochem Soc Trans. 2013;41:95-100
Hilgemann DW, Fine M, Linder ME, et al. eLife. 2013;2:e01293
Fuller W, Reilly L, Fraser NJ, et al. Proc Natl Acad Sci U S A. 2014;111:17534-9
Biochemical Society Summer Studentship Report 2015
TIE proteins: Bacterial ester domains

Angus Lovely
Supervisors: Ona Kealoha Miller and Dr Uli Schwarz-Linek
Biomedical Sciences Research Complex, University of St Andrews, North
Haugh, Fife, UK
1. BACKGROUND
In order to colonise their hosts, many bacteria, both commensal and pathogenic, use surface
proteins, such as microbial surface components recognizing adhesive matric molecules
(MSCRAMMs). Gram-positive bacteria show a diverse range of adhesin proteins. Many of
these are now known to contain intramolecular cross-links between amino acid residues,
which are self-generated upon folding. There are three main types of domains containing
such cross-links, isopeptide, thioester and ester domains (TIE domains)1. These are found in
clinically relevant bacteria such as Staphylococcus aureus and Streptococcus pneumoniae.
Isopeptide domains are known to be highly stable due to their intramolecular amide bond,
allowing the bacterium to have extreme resistance against mechanical stress. Thioester
domains mediate the covalent binding of bacteria to a host nucleophile via a reaction through
the thioester bond, resulting in a stable intermolecular bond2.
The existence of the ester domains in Gram-positive surface proteins is a recent discovery,
and it is not clear what the role of the domains is. Current evidence shows that they may be
linked to stability3, similar to the isopeptide domain, although it is possible that the bonds
susceptibility to nucleophilic attack, similar to thioesters, makes the domain suitable to play a
role in covalent interactions.
Figure 1: Examples of TIE proteins
from
Clostridium
difficile,
Clostridium
perfringens
and
Streptococcus pneumoniae.
2. KEYWORDS
MSCRAMMs TIE proteins thioester atomic force microscopy ester domains cross-links
3. GLOSSARY
SaED: this is the first ester domain from the Staphylococcus aureus SaTIE protein.
;
PnED: this is the first ester domain from the Streptococcus pneumoniae PnTIE protein.
Immobilised Metal Ion Affinity Chromatography (IMAC): this is a process of protein
purification; the protein of interest has a HisTag, a histidine rich repeat that has a high
affinity for a nickel ion column, which separates it from non-HisTagged molecules.
HisTag: A peptide containing six consecutive histidine residues. The His sidechain has a high
affinity for nickel (and cobalt) ion resins with high affinity allowing for quick and efficient
separation.
TEV protease: this is a protease isolated from the tobacco etch virus (TEV) that cleaves at a
specific sequence. It is used to remove HisTags from proteins of interest in reverse IMAC. The
TEV protease used is itself HisTagged, so can be removed from the protein mix along with the
cleaved HisTags.
Atomic Force Microscopy (AFM): a technique used to measure the strength of protein folds
proteins are attached to a cantilever via a silicon crystal and pulled apart whilst the force is
measured using a precise laser.
Site Directed Mutagenesis: a technique that introduces desired point mutations into amino
acid sequences, by selectively altering codons.

4. AIMS
Angus Lovely
The aim of the studentship was to target conserved residues of both a S. aureus and S.
pneumoniae ED protein (SaED and PnED) by site-directed mutagenesis (SDM) in order to
better the understanding of the mechanism of ester domain formation. Reconstitution
experiments were also carried out using variants of the PnTIE protein: PnoED (open ester
domain) and PnsED (split ester domain) using a PnET (ester tag) to observe if the ester bond
forms in trans in solution. The PnET is a short C-terminal peptide from the PnED fused to a B.
anthracis TIE protein thioester domain, BaTED. This experiment is being trialled as a similar
reaction has been observed for a split isopeptide domain from Streptococcus pyogenes4. This
reaction is so reliable that it is being used as a protein engineering technique. Finally, atomic
force microscopy (AFM) constructs will be designed. This will place the gene of interest into a
specially designed vector (pET3) that will express multiple copies of the protein of interest in
between IgG domains. This will allow the strength of the protein fold to be measured.
5. DEPARTURES FROM THE ORIGINAL PROPOSAL
There were no departures from the original proposal.
6. MATERIALS AND METHODS
Gel Electrophoresis: All SDS-PAGE gels were precast, manufactured by BioRad and were
made up of 12% polyacrylamide. The gels were run in SDS-Glycine running buffer.
Coomassie brilliant blue stain was used to visualize SDS-PAGE gels. The agarose gels were
cast in-house, and were all a 1% agarose solution in TBE. The gels were visualized with SYBR
Safe stain.
Side Directed Mutagenesis (SDM): This used to change single amino acids, by mutating
codons in DNA. Primers are designed that are complementary to the template DNA except for
the codon that is to be altered. After the initial polymerization stage, parent strand DNA will
contain the mutant codon, so will be amplified. A Dpn1 digest is performed afterwards to
remove any non-mutant DNA. Primers should be GC-rich at the ends to maximize primer
annealing.
Miniprep, Gel Extraction and PCR Clean-up: These three DNA purification techniques were all
performed using prepared kits. Minipreps and gel extraction were prepared using Sigma
kits and PCR clean-up using an Olympia kit.
Restriction Enzymes: Four different restriction enzymes are used in this project; SpeI,
BssHII, XhoI and NcoI. These are manufactured by ThermoScientific and are used in Buffer
Tango. The alkaline phosphatase used was also ThermoScientific.
Ligations: The DNA insert is in incubated with the plasmid vector in three different ratios,
1:1, 3:1 and 5:1. The DNA is incubated in d.H2O for annealing. A PCR machine is used to
reduce the temperature from 65oC to 4oC at a rate of 1oC.min-1. The annealed DNA is then
incubated overnight with T4 DNA ligase in T4 buffer.
Transformations: All transformations are carried out using pre-prepared agar plates.
Ampicillin and kanamycin are the only antibiotics used, both at a concentration of 1mM. 1L
of DNA is added to 50L of competent cells. The mixture is stored on ice for 30 min, heat
shocked at 42oC for 1min15s, returned to ice for 5 min, then ~250L of medium is added. LB
is the standard medium, and SOC medium is used for ligations. For ampicillin plates the liquid
medium is plated right away, and for kanamycin plates the medium is incubated first for an
hour, so the cells can grow and express antibiotic resistance (as kanamycin is a bacteriocide).
BL21 cells were used for expression, DH5 cells are used for DNA engineering (have no
protein synthesis apparatus) and SURE2 cells are used for DNA engineering where there are
lots of repeats (are missing certain replication proofreading apparatus). These are all
different cell lines of E. coli.
Test Expressions: Test expressions are carried out to determine the conditions in which a
protein is soluble. Cell line, medium, temperature, induction and buffer can all be altered. The
cell line used in all cases for this project is BL21, and the medium is LB broth. The cells are
grown up to an optimum density (OD600)=0.6, then are split into aliquots. The aliquots are
induced and left at the set temperature. ITPG is used to induce the cells, by activating the
lacI operon. After the set temperature, the cells are lysed by sonication, spun down, and the
Angus Lovely
soluble fraction is analysed by SDS-PAGE to determine if protein is present, by comparing

against the uninduced control. Test expressions are used to choose conditions for large-scale
expression. The buffer used for test expressions should have a pH more than one pH unit
away from the isoelectric point of the protein, so it remains soluble.
PCR/Colony PCR: Two different polymerase enzymes are used for PCR. Taq polymerase is
isolated from Thermus aquaticus and is thermostable up to 95oC, with an optimum
temperature of 75oC. Taq has no proofreading ability, but has an extension rate of 1 min.kb-1.
Pfu Polymerase is from Pyrococcus furiosus. It has proofreading capability and is
thermostable up to 100OC, and has an optimum temperature of 72oC. It has an extension
rate of 2 min.kb-1. Taq can be used for diagnostic PCR, but Pfu should be used for DNA
engineering. DNA is denatured at 95oC usually for 30s, annealing of the RNA primers occurs
at 5oC below the primer melting point, also usually for 30 seconds. Extension occurs at 72oC
for a time depending upon polymerase and amplicon length. DMSO can be added to the PCR
mix to prevent DNA secondary structure formation. Colony PCR is used to amplify DNA
directly from a plate. The PCR mix is made, a colony is transferred from one plate to another,
labelled, and then vigorously shaken with the PCR mix to transfer some of the DNA.
Plasmid Vectors: Two different vectors are used in the project. The SaED and PnED proteins
are both expressed in the pHisTEV vector. This vector includes a KanR gene, 6x His-tag, TEV
cleavage site, lacI operon and a T7 promoter region. XhoI and NcoI are used with this vector.
The AFM constructs are in the pET3 vector. This too has a T7 promoter region and a lacI
operon, but instead has an AmpR gene. SpeI and BssHII are used with this vector.
Protein Expression: Proteins are expressed in LB in volumes of 500mL in 2L flasks. Antibiotic
is added at a concentration of 1mM, and the broth is then inoculated with 2mL cells. The
flasks are then incubated overnight at a temperature determined by the test expression. The
culture is then spun down, and the pellet resuspended in a lysis buffer. The solution is then
passed through the cell disrupter, where the cells are lysed at 20 kpsi. The debris is then
filtered from the solution and the protein is purified.
Protein Purification: All of the expressed proteins used in this project have a His-tag and a
TEV cleavage site. The filtered protein mixture is passed through a Ni2+ column, which the
His-tag binds to. A weak solution of imidazole, which also has an affinity for Ni2+, is passed
through to prevent non-specific binding. Then, a strong solution of imidazole is used to knock
the protein of interest off the column. SDS-PAGE is used to identify the eluted fractions that
contain protein, which are pooled together. The imidazole is removed by dialysis, and TEV
protease is then used to remove the His-tag. The protein solution is passed back through the
column, and the cleaved His-tag, the His-tagged TEV protease and any leftover imidazole
bind to the column and the pure protein is eluted.
7. RESULTS OF RESEARCH AND OUTCOME OF STUDENTSHIP
7.1 Staphylococcus aureus Ester Domain (SaED)
The wild type SaED DNA was transformed on kanamycin-agar, two of the colonies were
cultured overnight, and DNA extracted and purified using commercial kits. This provided the
entire DNA stock to be used for all mutagenesis reactions of SaED. Primers were designed to
introduce two mutations into the SaED protein. Both mutations affect ester bond forming
residues; Thr-7 and Gln-125. A question was if a T7C mutant would now form a thioester
bond, and that the Q125E mutant would still form an ester bond.
Following the PCR to carry out the side directed mutagenesis, the PCR reaction product was
cleaned up, and the DNA was sequenced to confirm the mutation had been correctly
introduced.
The sequence was correct, so the two mutants were test-expressed to assess if soluble
protein can be obtained. The isoelectric point (pI) of the T7C mutant (with HisTag) is 6.43,
and 6.22 for the Q125E mutant (with HisTag). Therefore, PBS (pH 7.5) can be used for the
test expression. After the cells optimum density has been achieved and they have been
incubated overnight following induction, lysis was induced by sonication. Cells were sonicated
for three 15-second intervals. Cells were then spun down to remove debris, and both the
soluble and insoluble fractions were checked for protein content.
Angus Lovely
The test expression showed that the protein was highly soluble, and large-scale expression
was carried out. 25oC was chosen as the temperature for the overnight expression stage.
Following expression, the cultures were spun down to isolate the cells. These were
resuspended and lysed using a cell disruptor. The remaining solution was spun down again,
this time to remove cell debris and isolate the proteins in solution. The protein was purified
through IMAC, dialysed to remove the imidazole from the solution, and cleaved by TEV
protease and re-purified using IMAC to remove the HisTag. Gel filtration was then used to
further purify the protein eluted from this second, reverse IMAC.
Alongside this protein expression, another sample of both the mutants was prepared
differently. They were grown in minimal media (glucose, yeast nitrogen base and M9 salts) to
incorporate 15N into the cells through 15NH4Cl as the sole source of nitrogen. This isotope
labelling facilitates heteronuclear NMR spectroscopy.
The protein that was eluted was then concentrated to be analysed by various techniques. A
500M solution was used for NMR. A well-dispersed protein fingerprint (or 1H,15N-HSQC)
spectrum with the a number of resonances reflecting the protein sequence would be expected
for a protein with a well-defined fold, such as one containing an ester bond crosslink. As
shown in figure 2, the spectra obtained for both mutants contained many broad signals,
which is an indicator that the proteins were not fully folded, indicating that ester (or
thioester) bonds are not present in these proteins.
Figure 2: HSQC spectra for SaED WT, T7C and Q125E mutants. Clearly, the spectrum for the WT is
better defined than for the mutants, indicative of more structural rigidity in the protein.
Another piece of evidence to suggest that the mutants did not contain cross-links comes from
the mass spectrometry data. Figure 3 shows that the molecular weight observed for the two
proteins is 17025.10 Da (T7C) and 17024.10 (Q125E). This is the expected molecular weight
for the protein lacking the N-terminal methionine. If the thioester/ester bond was present
then the molecular weight would be 17 or 18.0 Da lower, accounting for the loss of an
ammonia or water molecule, respectively.
Angus Lovely
Figure 3: Mass spec of SaED T7C and Q125E mutants. Molecular mass of most abundant species shown
to be 17024.1 Da and 17025.1 Da respectively. There is a secondary species lacking 18 Da in both
spectra.
However, a secondary species is clearly visible in both spectra; these have a molecular
weight of 17006.6 Da (T7C) and 17007.7 Da (Q125E) less than the parent species. These are
the correct molecular weights for the thioester/ester bond containing protein. This indicates
that although the thioester/ester bond does not form as the dominant protein species, it may
be capable of forming to some extent.
Crystal trials were set up for both proteins at a concentration of 30 mg/mL. This
concentration was chosen as it is the same concentration as was used to solve the crystal
structure of the wild type protein. After a wide crystal screen was narrowed down, the ideal
conditions for crystallisation of the Q125E mutant were found to be 0.1M sodium acetate pH
5.0, 0.2M ammonium sulphate and 25% (w/v) PEG 4000. Crystal streak seeding with a cat
whisker was essential for the optimisation of crystal growth. Figure 4 shows the crystal
structure of this protein. An ester bond is not present, and does not form upon crystallisation
of the protein. This is interesting, as the H117A mutant of the SaED protein does form the
ester bond in crystal, although there is no ester bond present in solution. This difference may
be due to the fact that the His-117 residue is catalytic but non-essential, and not bond
forming like the Gln-125 residue. It appears that the chemical environment of the ester bond
is finely tuned to only allow ester formation form an amide (Gln) but not an acid (Glu). This is
in marked contrast to isopeptide domains.5
Figure 4: X-ray diffraction pattern and solved crystal structure for SaED Q125E mutant. Spacegroup
is P212121 and the resolution is 2.2 .
The fact that the ester domain lacking the ester bond is very similar in structure to the
wildtype indicates this bond is not a determinant of structure. However, the NMR spectrum
for the Q125E mutant shows the opposite in solution, as the protein fold is far less defined in
Angus Lovely
the non-bond containing mutant protein versus the wildtype.

7.2 Streptococcus pneumoniae Split Ester Domain (PnsED)
The second protein used for this project is PnED. The first part of the PnED project is the
generation of a split ester domain (PnsED). This is the PnED protein with the 12 C-terminal
amino acid residues, containing the ester-bond forming Gln, missing. The other protein used
is the ester tag (PnET). This is the final 11 C-terminal amino acid residues attached to the
BaTED protein (simply a choice of convenience, any other fusion protein would have been
suitable).
The first step in this project is to amplify the specific oED components (oED and tag), and to
include restriction enzyme sites, so that it can be inserted into an empty pHT vector and
expressed. The wild type PnED DNA was transformed, cultured overnight, isolated and
purified. The DNA was then treated with XhoI and NcoI. An empty pHT vector was also
treated with XhoI and NcoI, as well as alkaline phosphatase, to create stick ends to allow the
insert to anneal. The addition of the BaTED tag onto PnET required a 60nt long specialized
primer, in order to join the two peptides. Two separate ligations were then carried out to
create the PnsED protein and the PnET protein.
Once these new genes had been inserted into pHisTEV, the two separate proteins were testexpressed for solubility. Suitable conditions were found for large-scale expression, and the
protein was purified. The two separate proteins were then concentrated to be of roughly
equal concentration for reconstitution experiments.
Reconstitution experiments were carried out to attempt to bring
together PnsED and PnET in solution. This was done at protein
concentrations of 7 mg/mL and 5 mg/mL respectively, and each
experiment contained 21 g of protein in PBS. Different conditions were
trailed for the reconstitution experiments, and a time course was
carried out. Aliquots of the mixture were removed after 0 minutes, 15
minutes, 60 minutes and the remainder was left overnight. The
different experiments were separated by SDS-PAGE (figure 5),
alongside the individual parent proteins. The majority of the parent
protein is seen as an intense band lower down on the gel. The fainter
Figure 5: Positive
results of PnsED/PnET
bands in the parent lanes (to the left) represent dimerization of the
reconstitution
BaTED tag. There is some dimerization observed in the experimental
lanes (to the right), but also a small amount of what might correspond
to combined split ester domain and ester tag (highlighted in Fig. 5). However, there is also a
large amount of non-reconstituted protein in solution. The conditions in which the
reconstitution occurred were pH 8.0 and 9.5 incubated at 37oC for 1 hour in PBS.
The previous experiments showed that a small amount of protein was forming a cross-link in
solution, although this was only a small amount. It is possible that this is due to the missing
Asp-144 residue that is not present in either the PnsED or the PnET. The adjacent aspartate
is known to have catalytic properties, so it is entirely possible that the lack of success is due
to the missing residue. A primer was designed so as to insert an additional codon into the
PnsED gene to add the additional aspartate. This DNA has been sequenced, and the correct
result has been confirmed. It is currently in the test expression stage of large-scale protein
expression.
7.3 Streptococcus pneumoniae Open Ester Domain (PnoED)
The open ester domain (oED) is derived from the wild type PnED protein, with a single SDM
reaction carried out. It is Gln-146 close to the N-terminus that forms the ester bond in this
protein. The glutamine has been removed by SDM, and replaced by a small, non-conservative
alanine residue. The Q146A mutant does not form the ester bond (MS evidence). It is
thought that by mixing the PnoED with the PnET used with the PnsED will result in the tag
inserting itself into position to form the ester bond and disrupting the fold of the PnoED. This
is because without the ester bond, the fold of the WT PnED will be weakened.
The PnoED went through a process of mutagenesis, sequencing and test expression very
similar to the SaED mutants and the PnsED. Once the correct sequence had been confirmed,
and soluble protein conditions had been found, the protein was expressed, purified and
Angus Lovely
concentrated.
The ester bond is typically slow to form, but is very stable. Therefore, it was decided to trial
high temperatures for ester bond formation with the oED. Two different temperatures were
used, 50oC and 37oC at pH 7.5 and 9.5. On the gels shown in Fig. 6 there are experimental
and control lanes. The controls contain the PnoED and BaTED without the ester tag. This is to
ensure that any interaction between PnET and PnoED is due to the ester tag and not any
region of the BaTED protein. Aliquots of the experiment were removed and examined by
SDS-PAGE after 1 hour and 24 hours.
Figure 6: SDS-PAGE analysis of PnoED + PnET after 1 hour and 24 hours.
As can be seen in figure 6, the parent proteins are capable of dimerising similarly to the
PnsED. In the experimental lanes (5-8) there are some bands seen that are not present in
either the parent lanes (1-3) or in the control lanes (9-12). However, I believe that these
bands are of too great a molecular weight to represent an ester bond-containing complex
they are more likely to represent multimers of the BaTED protein.
7.4 AFM Constructs
The starting point for the AFM project was a plasmid inserted in the pET3 vector (named AFM
SRU); four repeating IgG domains are encoded with a single PnED domain in the vector after
the first IgG domain. IgG is included in AFM constructs as it is a well-studied protein that has
a well-defined and well-measured protein fold. It is used as a control when carrying out AFM
measurements. To accurately measure the strength of a protein there must be at least two
repeats of the domain in the construct.
The first step of this project was to isolate a new
PnED domain to insert into the pET3 backbone
(figure 7). An overnight expression culture was
made from a glycerol stock, and the DNA was
then isolated and purified. The DNA was then
digested with SpeI and BssHII in a sequential
digest. The SpeI digest was carried out first,
Figure 7: Initial AFM construct containing
followed by a BssHII digest. The buffer
four IgG domains and one PnED domain,
concentration was doubled between digests to
showing restriction sites.
maximize enzyme. The vector was also digested
with the same enzymes. This removed the third IgG domain where the PnED domain is to be
inserted, and also created sticky ends, so the PnED domain can anneal. The vector was also
digested with alkaline phosphatase, to remove the phosphate groups, so phosphodiester
bonds can form. After the annealing time, the new DNA was ligated into place.
Following the digestions and ligations, the DNA was prepared for sequencing to confirm that
the ligation was successful. Following this transformation, the DNA was transformed in SURE2
cells on an ampicillin plates, and then cultured overnight in order to make glycerol stocks.
The new AFM construct (named AFM SRU-2) was then test-expressed in Tris (pH 8.5) and
HEPES (pH 7.0). Large-scale expression was then carried out. Mass spec data is awaited to
confirm that the construct has expressed as expected.
Angus Lovely
8. FUTURE DIRECTIONS
8.1 SaED
Following the partial success of the attempts to observe a thioester bond in the SaED T7C
mutant, further experiments will be carried out to try and find conditions that favour thioester
bond formation. This will include adding DTT or BME, as the presence of a reducing agent will
prevent disulphide bridge formation, therefore, the cysteine residue will not be already
bonded if ester domain formation turns out to be slow. It will also involve using a higher pH
to favour base-catalysed [thio]ester bond formation.
The SaED T7C mutant will undergo crystallization screening in an attempt to grow a crystal
that is suitable for X-ray diffraction analysis. Therefore, this protein structure can be solved
by X-ray crystallography, as with the Q125E mutant. It will be interesting to see if an ester
bond forms upon crystallization as with the H117A mutant, which will be different to the
Q125E mutant.
8.2 PnsED
Conditions have been discovered in which the PnET and PnsED proteins form an ester bond in
solution (pH 8.0, 9.5 at 37oC in PBS). With the second split ester domain (PnsED-2)
synthesised containing the additional Asp-144 residue, it is thought that further reconstitution
experiments will show more promising results.
8.3 AFM Constructs
If the mass spec data for the AFM SRU-2 construct is correct then it can be used for collecting
atomic force microscopy data. The next stage will be to insert mutant PnED protein into the
AFM backbone. This will be done one domain at a time. First, the wild type PnED domain will
be replaced, and then the third IgG domain will be replaced, using digests and ligations. Once
the PnED T21A mutant is repeated in the AFM backbone, then this protein can also be
expressed. Then the data collected by AFM, the strength of the ester domain in the protein
can be calculated.
9. HOW GRANT HAS CONTRIBUTED TO MY CAREER ASPIRATIONS
The grant from the Biochemical Society has made me consider more seriously a career in a
lab setting, as I have enjoyed working significantly longer hours in the lab than I have
previously. It has also made me wish to pursue a PhD in a related field to my summer
studentship. Furthermore, this studentship has made me wish to explore the molecular
biology side of my biochemistry degree more, as this is a subject area that I have found most
interesting.
10. VALUE OF STUDENTSHIP TO MYSELF
This studentship has taught me a wide range of technical skills well beyond the scope of a
normal biochemistry degree. It has also given me a lot more confidence for the longer lab
sessions that I will be starting in the honours years of my degree. This has also given me an
excellent starting point for my honours project, and has also been influential in my applying
for an industrial placement next year as part of the masters degree.
11. VALUE OF STUDENDSHIP TO THE LAB
This summer project made an important contribution to an emerging project in the lab. Of all
cross-link containing protein domains that have been discovered over the past decade, ester
domains remain the least understood, and require investigation because they may harbour
important functions. The data obtained thanks to the support by the Biochemical Society,
while not conclusive in all cases, will support a grant application (BBSRC) and inform more
experiments. The construct made for AFM experiments will initialise a new collaboration (with
David Brockwell, Astbury Centre Leeds), and will open an entirely new angle in our research.
In addition to kick-starting a new project in the group and the scientific value, the summer
studentship also benefitted the supervisor in the lab, a 1st year PhD student, who found the
supervision experience very valuable, and rewarding.
Angus Lovely
12. REFERENCES
1. Schwarz-Linek U, Banfield MJ (2014) Yet more intramolecular cross-links in Gram-positive
surface proteins. Proc Natl Acad Sci 111:1229-1230
2. Walden M et al. (2015) eLife 4:06638
3. Kwon H et al. (2014) Proc Natl Acad Sci U S A. 111:1367-1372
4. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M (2012)
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial
adhesin. Proc Natl Acad Sci 109:690-697
5. Hagan R et al. (2010) Angew Chem Int Ed Engl 49:8421-8425
13. NOTE
The X-ray diffraction pattern, and the solved crystal structure in figure 4 are unpublished
data. Please do not share without permission.
The Characterisation of Calcium Response and Neuron-Specific Markers on Different

Cell Lines for Parkinsons Disease Modelling
Student: Azilleo Kristo Mozihim, University College London
Supervisor: Dr. Michael Duchen, University College London
Background
Parkinsons Disease (PD) is a debilitating neurodegenerative disease marked by the loss of
dopaminergic neurons (DA) in the substantia nigra (SN) of the midbrain. Molecularly, it is marked by the
pathological presence of abnormal intracellular aggregates of misfolded protein, notably -synuclein, called
Lewy bodies (LB). Clinically, it is diagnosed based on the presence of four characteristic motor symptoms:
rigidity, bradykinesia, resting tremors and postural instability. PD has an estimated prevalence rate of 0.3% in
the industrialised nations and about 127,000 sufferers in the UK [1]. Thus, considering the debilitating
symptoms and significant number of sufferers, substantial investment has been made into PD research to
develop effective treatment for it. However, before treatments can be devised, it is imperative to gain in-depth
understanding of PD and one of the ways is firstly to develop an accurate disease models of PD, one of which
could be a cellular disease model. This type of model allows investigators to understand a particular aspect of
PD and different cellular models using different cell lines are used to investigate different aspects of PD. For my
project, I investigated the suitability of three cell lines primary neurons, SH-SY5Y and neural progenitor cells
(NPC) to be used as PD cell models for studying cellular calcium response in PD. Additionally, I also
investigated the presence of neurone-specific markers MAP2 and NeuN- to determine the extent to which SHSY5Y and NPC are similar to primary neurons.
Methods
Calcium Response Assay
A coverslip with the cell samples adhered on to it were incubated with 300 L of calcium dye (5 M of
either Fluo-4 or Fura-2) at 37oC for 30 minutes.
The calcium dye was removed and replaced with 500 L of recording buffer (150mM NaCl, 4.25mM
KCl, 4mM NaHCO 3 , 1.25mM NaH 2 PO 4 , 1.2mM MgCl 2 , 1.2mM CaCl 2 , 10mM D-glucose, 10mM
HEPES, pH 7.4)
The cell samples then were subjected to fluorescence microscopy with the settings depending on the
dye used. For Fluor-4, the excitation wavelength was set at 488 nm while for the Fura-2, two channels
were used with different excitation wavelength: 340 nm and 380 nm.
After a few minutes of frame acquisition, the 100 L of the stimulus was added with final
concentration of 100M ATP, 10M glutamate or 50M potassium solution.
If the dye used was Fluor-4, 100L of 2M (final concentration) ionomycin was added after a few
minutes of stimulus addition.
The images were analysed using ImageJ and Microsoft Excel.
Detection of Neuron-Specific Markers
The cell sample were fixed with the relevant antibodies, that is, either Abcam Anti-MAP2 antibody
(ab11267) or Milipore Anti-NeuN Antibody (MAB377) at 1:1000 dilution and Invitrogen Alexa Fluor4 anti-mouse IgG as the secondary antibody at 1:4000 dilution.
The fixed sample were then subjected to fluorescence microscopy using with the excitation wavelength
set at 488 nm.
Results and Discussion

Characterisation of Stimulus-Evoked Ca2+ Response in Primary Neurons, SH-SY5Y and NPCs.
For glutamate (Figure 1), the primary neurons exhibited the expected calcium response, that is, a sharp
increase in intracellular calcium concentration (ICC) upon glutamate addition followed by a gradual decrease
until it levels at an above-baseline concentration. The SH-SY5Y, on the other hand, did not exhibit any response
upon glutamate addition and this is inconsistent with that reported by previous investigators [2]. This might due
to the difference in glutamate concentration used: previous investigators used 500 M whereas 10 M was used
for this experiment. For the NPC, upon glutamate addition, there is a sudden decrease ICC followed by its
sudden rise which then levels at a below-baseline level. Such a response is inconsistent with that reported by
previous investigators and this most likely due to the differences in the NPC culturing conditions used for this
experiment and those used by the previous investigators [3].
For ATP (Figure 2), the primary neurons exhibited the expected response, that is, a gradual increase of
ICC followed by its gradual decrease. For the SH-SY5Y, there was a sharp increase in ICC upon addition
followed by a sharp decrease which then levels at baseline level. This is a similar response obtained by previous
investigators, albeit at a smaller magnitude which most likely reflects the difference in the concentration of ATP
used [4]. For NPC, the calcium response is erratic (i.e. the ICC continuously decrease and increase, forming a
zig-zag like pattern). Even so, it seems that the changes of the ICC is centred about a mean. This response is
unlike the one reported by the previous investigators and, again, it most likely reflects the differences in the
NPC culturing conditions [3].
For the high potassium solution (Figure 3), the primary neurons, the ICC increased gradually until it
reaches a peak and then gradually decreases to level at an above-baseline level, the response consistent with
previous investigators. For the SH-SY5Y, the ICC increased sharply and the ICC levels at that the increase level,
consistent with the response obtained by previous investigators [5]. For NPC, no calcium response assay with
high potassium concentration could be done due to time constraint and lack of viable cell sample.
MAP2 Antigen Detection
For primary neurons, there is clearly the presence of MAP2 which are located on the axons and cell
bodies. For the NPC during the 3rd passage, there are distinct near-circular objects visualised, suggesting that
these are MAP2-expressing NPC. However, for the NPC during the 11th passage, shadows of what appears to be
neurone-like cells are observed. This could be due to the failure of the permeabilisation step during sample
preparation, resulting in the inability of the MAP2 antibodies to enter the NPC and attach to MAP2. Even so,
considering that the 3rd-passage NPC express MAP2, it is plausible that the 11th-passage NPC too express them
but only a repetition of the MAP2 detection step can confirm this. For both NPC, there was significant amount
of background staining which was mostly likely due to insufficient amount of washes with the phosphate buffer
solution (PBS) during the sample preparation stage (Figure 4)
NeuN Antigen Detection
For primary neurones, near-circular shapes can be visualised, suggesting the expression of NeuN as
expected. Similarly, the 3rd-passage NPC shows many small circular objects evenly spread, suggesting that they
too express NeuN. For the 11th-passage NPC, NeuN is detected surrounding what appears to be cell bodies,
indicating that these cells express NeuN as well (Figure 5).
Future Directions
Conducting the calcium response assay on NPC with high potassium solution as the stimulus could
provide further information regarding the calcium response of NPC. Additionally, the NPC used could be
cultured in the lab wherein the experiment is conducted instead of obtaining them from another lab to ensure
proper and consistent culturing conditions. Besides that, additional experiments can be conducted to convert the
fluorescence data to actual intracellular calcium concentrations. Each part of the experiment could be repeated
to gain additional data and to obtain higher quality particularly for those with data that are compromised due to
technical difficulties experienced. Finally, the iPSC can be subjected to the same experiments to assess its
suitability as a cell model for calcium response modelling of Parkinsons disease.
Departure from original proposal
Ideally, each of the calcium response assay should be the done with only one type of dye but two
different type of dyes had to be used due to the malfunction of one of the fluorescence microscopes during the
experiment. Experiments should have been done on iPSC as well but this was not possible due to inability to
timely acquire the samples.
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Figure 1 The Intracellular Calcium Response of the Cell Samples towards Glutamate
10 M glutamate was added at the time indicated by the first arrow for each cell sample. In each graph, each
series line represents the intracellular calcium response of a single cell. The difference in the labelling of the yaxis reflects the different type of dyes used: fura-2 was used for the first two from the top while fluor-4 was used
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Figure 2 The Intracellular Calcium Response of the Cell Samples towards ATP
100 M glutamate was added at the time indicated by the first arrow for each cell sample. In each graph, each
series line represents the intracellular calcium response of a single cell. The difference in the labelling of the yaxis reflects the different type of dyes used: fura-2 was used for the one at the bottom while fluor-4 was used for
first two from the top. For fluor-4, a second arrow indicates the addition of 2 M ionomycin.
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Figure 3 The Intracellular Calcium Response of the Cell Samples towards High Potassium Solution
50 M of potassium solution was added at the time indicated by the first arrow for each cell sample. In each
graph, each series line represents the intracellular calcium response of a single cell. For both cell samples, fura-2
was used as the calcium dye.
Figure 4 Expression of MAP2 in, from left to right, primary neurons, 3rd-passage NPC and 11th-passage
NPC
Figure 5 Expression of NeuN in, from left to right, primary neurons, 3rd-passage NPC and 11th-passage
NPC
References
1.
de Lau, L.M. and M.M. Breteler, Epidemiology of Parkinson's disease. Lancet Neurol, 2006.
5(6): p. 525-35.
2.
Naarala, J., et al., Excitatory amino acid-induced slow biphasic responses of free intracellular
calcium in human neuroblastoma cells. FEBS Lett, 1993. 330(2): p. 222-6.
3.
de Groot, M.W., et al., Characterization of calcium responses and electrical activity in

differentiating mouse neural progenitor cells in vitro. Toxicol Sci, 2014. 137(2): p. 428-35.
4.
Larsson, K.P., A.J. Hansen, and S. Dissing, The human SH-SY5Y neuroblastoma cell-line
expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel
function. J Neurochem, 2002. 83(2): p. 285-98.
5.
Brater, M., et al., Voltage-sensitive Ca2+ channels, intracellular Ca2+ stores and Ca2+release-activated Ca2+ channels contribute to the ATP-induced [Ca2+]i increase in
differentiated neuroblastoma x glioma NG 108-15 cells. Neurosci Lett, 1999. 264(1-3): p. 97100.
Determining the phospho-specific interactions of 53BP1 and their role in DNA damage
repair
Student: Caitlin McCarthy, University of Sussex
Supervisor: Dr Felicity Watts, University of Sussex, Genome Damage and Stability Centre G4.08
Background
When DNA double strand breaks occur, the cell has mechanisms to recognise and fix the damage. An MRN complex
firstly finds and binds to DSBs. This complex then goes on to recruit ATM, a kinase which phosphorylates
downstream proteins such as H2AX. When histone H2AX is phosphorylated, MDC1 is recruited and causes more
MRN and ATM to be recruited which amplifies the signal. H2AX also causes histone modification to allow proteins to
have better access to the site of damage.
An important protein recruited to the DSB is 53BP1. 53BP1
is a large protein with many different domains (Fig 1). It
has a BRCT domain pair at the C-terminus that has a role in
repair of damage in heterochromatin. The central part of
Fig 1 Organisation of 53BP1
the protein has several sites which allow the protein to bind to the damaged
chromatin and help form foci. The N-terminus of 53BP1 is large and contains many
serine and threonine residues that can be phosphorylated and then go on to recruit other proteins to the site of
damage.
53BP1 has an important role in the G1 phase of the cell cycle
as it promotes canonical Non Homologous End Joining
(cNHEJ) when DNA damage occurs during this phase. cNHEJ is
an easy way of fixing the break as it involves simply joining
the DNA ends together. It is also believed that 53BP1 may
have a role in the G1 damage checkpoint to arrest cells in G1
until the damage has been repaired.
Previous work from our lab has shown that in vitro, 53BP1 colocalizes with TopBP1 at DSBs. 53BP1s N-terminus interacts
with BRCT domains of TopBP1: specifically, BRCT4,5 binds to
phosphorylated Ser366 on 53BP1 and BRCT1,2 binds to
phosphorylated Thr670 (data not shown). in vivo 53BP1-S366A
and -T670A mutations result in an inability of 53BP1 to colocalize with TopBP1 (Fig2). In addition, it has been shown that
S366 and T670 are phosphorylated in vivo when the DNA is
damaged (e.g. Fig 3A).
DAPI
HA-53BP1 TopBP1
CENPF
Merge
WT
S366A
T670A
Fig 2 53BP1 S366A and T670A mutations affect colocalisation of

53BP1 and TopBP1. U2OS cells were reverse transfected with
53BP1 siRNA, 48 h later cells were transfected with an siRNA
resistant construct containing wt 53BP1 or S366A or T670A
mutants. 16 h later cells were exposed to 8 Gy IR. Cells were left
to recover for 4 h before staining for HA, TopBP1 and CENPF (to
identify G1 cells that dont stain with CENPF). Cells were
visualised using a Deltavision spectris microscope.
Aims
To identify the kinases responsible for the phosphorylation of Ser366 and Thr670 in response to damage.
Materials and Methods
Tissue cell culture, transfection, irradiation and inhibition of kinases

Hela cells were grown in DMEM media. These cells were then split. Cells were reverse transfected with siCont, while
others were transfected with 53BP1 siRNA. This was done by plating out 2x105 cells on dishes and adding 50l
Optimem and 5l of either siCont or si53BP1. Cells transfected with siCont or 53BP1 siRNAs were irradiated 48 h
after transfection. One h after the inhibitor was added, cells were irradiated with 8Gy IR using a 137Cs source. Kinase
inhibitors were added to appropriate final concentrations (caffeine 20 mM, Roscovitine 50M, SB203580 MAPKi
10M and CKIIi TBB 10M).
SDS page and western blot
Whole cell extracts were prepared from cells using standard protocols. Samples were boiled for 5 min and spun at
13,000 rpm for 5 min before being loaded and run on a 7.5% SDS polyacrylamide gel. The separated protein was
then transferred onto a PVDF membrane. Specific antibodies to peptides containing the regions surrounding
phosphorylated S366 or phosphorylated T670, 53BP1 (to check knockdown) and tubulin (loading control) were then
added in 4% milk to each membrane and left on a shaker overnight. The primary antibody was washed off and the
secondary antibody conjugated to HRP was then added to each membrane. Once the secondary antibody has been
left for an hour, unbound antibody was washed off and the membranes were soaked in light and dark solution ECL
reagent and exposed to X-ray film.
Results and Conclusions
Western analysis with phospho-specific antibodies against pS366 and pT670 peptides of extracts from cells +/- siRNA
53BP1 and +/- IR indicate that 53BP1 is phosphorylated in vivo in response to IR (Fig 3 compare lanes A1, B1 with A3,
B3). We wished to identify the kinases responsible for phosphorylation of S366 and T670. Computer analysis showed
that cdks, MAPK and CK2 as the most likely candidates. Cdks were first to be tested using Roscovitine which inhibits
most cdks. Comparison of Fig 3A,B lanes 3 and 5 indicate phosphorylation of S366 and T670 even when cdks are
being inhibited. Thus cdks do not appear to be the responsible kinases. In Fig 4A,B lanes 3, caffeine was added as a
control as it inhibits all kinase activity. A decrease in pS366 and pT670 can be observed in these lanes compared to
lanes 1. In lanes 5, there is phosphorylation of S366 and T670 even when MAPK is being inhibited, suggesting MAPK
is also not the responsible kinase. However, in this experiment DMSO, the solvent for the inhibitors, appears to
affect phosphorylation as no bands are visible in lanes 4, thus this experiment needs to be repeated. Fig 5 shows in
lane 5 that CK2 inhibition also has no effect on phosphorylation of Ser366. Therefore neither cdks, MAPK or CK2 are
responsible for the phosphorylation of Ser366, and cdks and MAPK are not the kinases for Thr670.
Fig 3 CDKs do not phosphorylate S366 or

T670. Hela cells were transfected with
either siRNA targeting 53B1 or a nontargeting control, followed by treatment
with Roscovitine (CDK inhibitor). Cells were
137
irradiated with 8Gy using a Cs source 4h
before lysis. Cell extracts were separated by
SDS PAGE and western blotted with antisera against pS366, pT670, 53BP1 and
tubulin
Fig 4 MAPK does not phosphorylate

S366 or T670. Hela cells were
transfected with either siRNA targeting
53B1 or a non-targeting control,
followed by treatment with caffeine
(inhibits all kinase activity), DMSO
(solvent for inhibitors) or MAPK
inhibitor. Cells were irradiated and
processed as in Fig 3.
Fig 5 CK2 does not phosphorylate S366. Hela

cells were transfected with either siRNA
targeting 53B1 or a non-targeting control,
followed by treatment with caffeine, DMSO or
CK2 inhibitor. Cells were irradiated and
processed as in Fig 3.
Future Directions
Future work is to continue looking for the kinase responsible for the phosphorylation of S366 and T670 in response
to damage. Once the kinase has been determined, the role of the phosphorylation-mediated interaction with
TopBP1 will be determined, e.g. the effect on repair of DSBs in G1 and G1 damage checkpoint.
Departures from the Original Proposal
The original project was to probe the role of the phosphopeptide binding site of the BRCT domain of 53BP1. Since
submitting the application this work was carried out by another member of the lab. I therefore undertook a different
project, but analysed the same protein (53BP1). In this new project I learnt a range a techniques such as being able
to split and transfect HeLa cells, visualisation of transfected cells using the spinning disk microscope and was able to
perfect my skills in SDS PAGE and western blotting. I have consolidated and refined my knowledge in molecular
genetics, cell biology and the DNA damage response and now feel much more confident in the lab.
Characterising mutants of -phosphoglucomutase

Biochemistry Society Summer Vacation Studentship
Carly Wynne
Supervisor: Angus Robertson - Biophysics, Department of Molecular Biology
and Biotechnology, University of Sheffield
-phosphoglucomutase (PGM) from Lactococcus lactis is a phosphoryl-transfer enzyme that has been the
focus of intense research for over a decade. PGM catalyses the interconversion of -glucose-1-phosphate
(G1P) to -glucose-6-phosphate (G6P), lying strongly in favour of the formation of G6P with an equilibrium
constant of 28 (2). The enzyme is a phosphomutase and catalyses both phosphatase and kinase reactions. PGM
has a mass of 25kDa and can exist in both open and closed states, which
alternate depending upon whether the forward or reverse reaction is
proceeding (3). The active site is located between a core and a cap
domain and is freely accessible to solvent when in the open conformation.
A catalytic Asp8 residue is found in the active site, with Asp10, Thr16,
Ser114, Lys145, Glu169 and Asp 170 all found within a 6 radius of this.
The Mg2+ cofactor is octahedrally coordinated by the backbone carbonyl
of Asp10, the side chains of Asp8, Glu169 and Asp170, one oxygen from
the Asp8 phosphoryl group and one water molecule. The phosphate
bound to Asp8 also interacts with the side chains of Ser114 and Lys145
Fig. 1 Diagram showing the Mg2+ octahedral
(4) (Fig. 1.) The coordination of this Mg2+ cofactor is crucial to the
geometry in the active site of PGM
functionality of the enzyme, by
balancing the negative charges of
several active site residues. The mechanism of PGM has continued to cause
fierce debate, especially regarding the structure of the transition state. This
debate intensified when it was claimed that a pentavalent phosphorane
intermediate was directly observed using high intensity x-ray crystallography
(2.4 resolution) and electron density maps, however this was later proved to
be an MgF3- molecule. Baxter et al determined that the electron density
observed in crystal structures of PGM were from an MgF3- molecule. The
group then used difference Fourier maps to explain the electron density
patterns observed with MgF3- compared to a PO3- molecule. The initial
proposal of a pentacovalent intermediate showed large difference peaks when
Fig.2 Diagram showing the
mapped in this format (Fig. 2A), however these could be resolved when the
difference Fourier maps for a
PO3- is replaced with MgF3- (Fig. 2B). MgF3- is isoelectronic with a PO3pentavalent
phosphorane
molecule, with both molecules possessing 24 valence electrons, a net negative
intermediate (A) and an MgF3charge, and the ability to form pentacoordinate geometry, and therefore
transition-state analogue (B) in
the active site of PGM (Baxter et
provides a way to observe a PGM transition-state analogue (TSA) (5). Due to
al, 2010)
the timescale of this reaction, it is not possible to directly observe the structure
as the reaction processes. Therefore, molecules that mimic the PO3- of the
substrate are used to observe the reaction at a certain point, including BeF3- and the previously mentioned MgF3-.
Due to the geometry of Beryllium (strictly tetrahedral and cannot form a pentacovalent structure), when added
to PGM, the Be(OH)3 and NaF form a covalent group that assembles on D8, forming a ground-state analogue
(GSA) of the enzyme. The electronic configuration of the BeF3- moiety prevents nucleophilic attack, thus
limiting the complex to a GSA and providing an elegant example of the importance of stereoelectronic effects,
originally highlighted by Blackburn et al (6).
A minor set of peaks are observed in the wild type fluorine NMR spectra, corresponding to a change in
coordination geometry of the transition state mimic Mg2+, via the addition of a carbonyl ligand from the protein
backbone (Fig.1). The rotation of this carbonyl is implicated in a gating mechanism, whereby the enzyme
prevents back reaction of the transferring phosphoryl group. K145A is a mutant that displays similar fluorine
chemical shifts and thus was a suitable candidate for further study.
Our Research
We will focus on characterising several mutants in an attempt to shed light on the minor conformation, which is
likely to be involved in intermediate release and enzyme catalysis, with the aim to determine the driving force
behind the inter conversion of the two species. Asp10 has been observed to move in and out of the active site
throughout the reaction, however D10 importance beyond the role as a general base is unknown. Lys117 is a
residue that assists the closing of the two domains when the substrate is bound in the active site. We aimed to
create two mutations, D10A and K117A, and observe the structure and function of the enzyme with these
changes. However, due to complications with equipment and sourcing the D10A/K117A mutants, our focus
shifted to Pro138 and K145 mutants of PGM.
Pro138 occupies two different environments in the apo structure, indicating cis-trans isomerism, which may be
responsible for the two species viewed in previous NMR experiments. Therefore, a P138A mutant was created,
which removes the capacity of the residue to occupy these two environments, and consequently may
validate/invalidate the hypothesis that isomerism is the driver behind the two species observed. Preliminary
NMR data has suggested that this mutant did not affect the ratio of the two species in the apo enzyme, however
the crystal structure will give more indication of how, if at all, this mutation changes the ratio of the two species.
We will characterise the mutant as an apo enzyme, and as a TSA using an MgF3- molecule. A crystal structure
of the wild type (WT) apo enzyme shows that this proline residue is within Van der Waals contact distance with
the carbonyl that rotates in and out in the minor/major transition, and therefore may be having an effect on the
ratio of the two species. Therefore, we aim to create crystals of the P138A TSA to see if this is the case, as in
WT crystals the minor conformation has not previously been observed.
Another mutant that we will focus on is K145A, a mutant that has previously been observed to exist in the
closed minor form as a crystal structure, when saturated with MgF3-. However, further characterisation using
NMR determined that, in solution, the mutant adopts a more open conformation and requires high
concentrations of sugar and metal fluorides to saturate the complex. Consequently, as aluminium has a higher
affinity for the enzyme-sugar than magnesium, AlF4- is used to form a TSA complex as opposed to MgF3-, as
energetic costs of an equilibrium between MgF3- and MgF2 push the complex away from forming a TSA. Our
aim is to form this TSA with AlF4- to analyse using NMR, and to obtain a crystal structure, as NMR does not
tolerate the high levels of MgF3- used to form the original crystal. Our final aim is to characterise the double
mutant K145A D10N. D10 may be involved in an activation step, as it is a general base that moves into the
active site to mediate catalysis. Previous analysis shows that D10N retains some catalytic activity before
forming a bisphosphate intermediate and inactivating itself. A crystal structure has already been obtained for a
D10N bisphosphate GSA, in the presence of the catalytic Mg2+, however, the NMR data showed significant
chemical shift changes between the Mg2+ bound and unbound states. Therefore, we need a crystal structure of
the D10N Mg2+ unbound state.
Methods & Materials

Competent Cell Preparation
Four E.coli cell lines (XL-gold, XL-blue, XL-blue SC and BL21-DE3) were plated on to LB plates, which were
incubated overnight at 37C. Two colonies from each of the original cell line plates were selected and inoculated
in 5 ml LB broth, and incubated overnight at 37C. A solution of LB was created and autoclaved along with four
conical flasks, ready for next step. 0.5ml of the cells were added to 50ml LB broth and incubated at 37C until
the OD reached 0.6 (exponential growth phase). The cells were then made competent in several steps. Cells
were centrifuged and resuspended in 10ml ice cold CaCl2, and incubated on ice for 10 minutes. Cells were then
centrifuged once more, and re-suspended in 2ml CaCl2 with 70l DMSO. Ice for 10 minutes, and further 70l
DMSO was added, and 200l aliquots were dispensed into freezer Eppendorf tubes. Stored at -80C. Check
competency of the four cell lines with a transformation of the WT plasmid. 2l of plasmid added to the cells,
and incubated on ice for 15-20 minutes. Heat shocked at 42C for 45 seconds, and then placed on ice for 2
minutes. 800l of LB (no amp) added and incubated at 37C for one hour. Plated onto LB agarose + amp plates
and those that have up-taken the WT plasmid formed colonies.
Mutagenesis
The D10A and K117A mutations were created by using mismatched forward and reverse primers to the wild
type plasmid. Solution of 70l prepared: 5l forward primer, 5l reverse primer, 5l WT plasmid, 5l 10x
reaction buffer, 1l PfuUltra HF DNA polymerase, 1l dNTP mix, and topped up with 28l H2O. These
plasmids encoded the mutation and an ampicillin resistance marker, allowing for selection when the PCR
products are plated. After 18-20 PCR cycles, WT methylated plasmids were digested with DPN1. The products
were plated on LB agarose + amp plates.
Transformation & Mini-Prep
Using the XL-blue SC and BL21-DE3 strains, a mini prep was prepared, by adding wild type plasmids to each
solution and plating. These were then incubated at 37C. Two colonies from the mini prep plates were selected
and inoculated in 10ml LB broth to maximise DNA mass. These plates were incubated at 37C overnight. The
QIAprep Spin Miniprep kit. 850l of mini prep solution was added to the column, and several buffers added
(including lysis and neutralisation buffers), in accordance with the kit instructions, before eluting the DNA using
sterile water. Both solutions were transferred to microfuge tubes and stored at -20C until required. WT and
K145A D10N plasmids were transformed into XL-Blue SC cells, and mini-prepped using the same kit and
protocol as previously listed.
X-ray Crystallography
X-ray crystallography experiments were set up to try and visualise mutants of PGM that have already been
isolated. A motherliquor of 700l was dispensed in the well, including PEG 4000 as the precipitant, 200mM
NaAcetate as the salt, and 100mM Tris pH 7.5 buffer. This was topped up with an appropriate volume of
sterilised water. Concentrations of 24%, 26%, 28%, 30% and 32% PEG 4000 were used. 2l of this
motherliquor was pipetted onto a cover slip, along with 2l of isolated protein. 4 mutants/complexes were used:
wild type (WT) apoenzyme, K145A-BeF3, K145A-BeF3-G6P and K145A-BeF3-G6P (half saturated and
saturated conditions). The crystals were viewed under a microscope, and one drop of the apo WT was selected,
and one drop of the apo K145A line. These crystals were used for a seeding experiment of the same 4
mutants/complexes, where they act as nucleation sites for further crystal development. The motherliquor was
prepared in the same way, however the crystallisation solution was made up of: 1l protein, 0.3l seeding
solution and 0.7l motherliquor. A further 7 mutants/complexes were laid: WT-Ammonium Sulfate, P138A
apo, P138A-BeF3, P138A-BeF3-G6P, P138A-MgF3-G6P, K145A apo, K145A-BeF3-G6P and a second stock of
K145A-BeF3-G6P (with 10mM G6P). These trials were carried out under the same conditions as previously
stated. Any needles/crystals that formed were extracted and used for seeding further experiments. A needle
pipette was used, which partially breaks up the crystals, and this solution was transferred into a microfuge tube.
The microfuge tube was vortexed for ~10 seconds. A serial dilution was performed, and the 100,000 fold
dilution used for seeding. K145A apo, P138A-BeF3 and P138A-BeF3-G6P mutants were relayed using seeding
solutions (same method as previously stated). K145A-AlF4--G6P was trialled under the same conditions as
previously stated, however, no crystals were observed. Buffer exchange of K145A-AlF4--G6P was performed
using 50mM BisTris pH 6.0 (5mM MgCl2 and 2mM NaN3), so the complex was now at pH 6.0. D10N MgF3trialled using conditions where crystals previously observed: PEG 4000 24-34%, NaAc 200mM and pH 7.5 Tris
buffer. This complex also trialled with seeding stock P138A BeF3- 10,000 fold.
NMR
NMR spectroscopy was performed across two institutions: University of Sheffield and University of
Manchester. Measure rate of turnover of PGM + 1M (NH)SO using NMR. Total solution of 500l including
50l PGM (1200nM), 14.1l AcP (20mM), 5l TSP, 45l D 2O and topped up with 364l H2O (16.67l 10mM
G1P added later). Initially, achieve 1D spectra with peaks for PGM, AcP and PPi. Enzyme now primed. Add
G1P and run NMR. Fluorine, proton and 2D TROSY NMR were performed on four complexes - K145A-AlFG6P, K145A D10N MgF3-G6P, P138A-MgF3-G6P and P138L-MgF3-G6P.
Results
Competent Cell Preparation
Analysis of the transformant plates showed that XL-Blue and XL-Blue SC had a high transformation efficiency,
whereas BL21-DE3 and XL-Gold showed slightly lower transformation efficiency with fewer colony growths.
This may be due to the plates reaching too high an O.D. (1.03 and 1.20 respectively), which results in cells
passing the exponential phase and dying after being transformed. Therefore, competent cell preparation was
repeated for XL-Gold and BL21-DE3.
Mutagenesis
The PCR product transformations plates had no colonies. This could be due to many factors, including the PCR
kit itself, incorrect primer sequences or missing out a certain reagent. The reason for lack of colonies will have
to be identified and the experiment repeated. The mutagenesis experiment is repeated with a different kit. The
PCR products are transformed into the competent cells, and plated onto LB + amp plates. This mutagenesis did
not work and two control mutagenesis reactions were performed (P138A and control solutions). These also did
not work and therefore this suggests that there may be an issue with the PCR kit not working correctly.
Transformation & Mini-Prep
There was no visible change in cell density in the starter culture for WT and K145A D10N transformants,
therefore this protocol is repeated using XL-Blue cells to determine whether the other cells were not efficiently
transforming. These new starter cultures produced WT DNA of approximately 100ng/l and K145A D10N
DNA of approximately 100ng/l.
A
B
X-ray Crystallography
The initial trials produced needles
A
B
for only K145A apo, P138A BeF3
and P138A BeF3 G6P, therefore
these are used to create seeding
solutions.
Those
mutants/complexes that produced
no needles/crystals were seeded
with seeding solutions of the same
mutant class. The seeding of the 4
mutants that initially produced no
crystals, yielded useable crystals
for P138A apo (Fig.3A) and P138A
Fig.3 P138A apo crystals (A) under conditions PEG 4000 23-32%, 200mM NaAc and 100mM Tris
MgF3 G6P and therefore seeding
buffer pH 7.5 and the electron density map of crystals observed at a resolution of ~1.7
solutions were created from these
crystals. However, despite seeding,
there were still no crystals for WT-Ammonium Sulfate (1M) and K145A BeF3 G6P. The WT-Ammonium
Sulfate (1M) will be tested using the robot to survey a range of conditions. The crystals were shot using the
Diamond synchrotron in Oxford. Electron density maps were formed with a resolution of ~1.7 and the
molecular replacement completed for the apo structure (Fig. 3B). By the end of my project, these were yet to be
analysed.
NMR
When PGM + (NH)SO was observed as a crystal
structure, only one species was isolated. This led to the
hypothesis that the (NH)SO may reduce the lag phase,
which was previously believed to be the enzyme being
primed, by having the enzyme already in the activated
state. However, a lag phase was still observed, suggesting
that the lag phase may not be accredited to a change in
enzyme conformation of the enzyme. Fluorine NMR for
the K145A-AlF-G6P complex was run at a pH range of
6.5-8 (Fig.4). The movement of the peaks highlighted
suggests that one species of this complex is pH dependant.
In this spectra, species are identified by four fluorine
peaks, attributed to the 4 fluorine atoms of the AlF
bound to the active site. Currently, there is no indication of
the structures of these two species are, and this would
require further analysis.
Fig.4 Fluorine NMR spectra of K145A-AlF-G6P at pH 6.5

(green), 7.0 (blue) and 8.0 (red)
The 2D TROSY experiments of P138A-MgF3-G6P (Fig. 5A)

and P138L-MgF3-G6P (Fig. 5B), in comparison to wild type spectra, have significant overlay, suggesting that
there is no change in folding state and that the TSA is very similar to the WT. However, it can be noted that the
overlay for the P138L TSA is not as good, which suggests that this mutation has a greater effect than P138A.
When these 2D TROSY spectra are overlaid, it is clear that there are little discrepancies in the overlap of the
peaks, with the tryptophan mutants in identical environments (Fig. 6A), however P138L has a smaller
tryptophan peak, suggesting that the complex is not saturated and there is a slow exchange between apo and the
TSA. Despite the differences in the 2D TROSY spectra, there is very little difference between the P138A-MgF3G6P and P138L-MgF3-G6P fluorine spectra (Fig. 6B). The Fa peak of P138a is downshifted, suggesting the
mutation has caused this fluorine to experience an increased magnetic field due to a decrease in shielding by
neighbouring atoms, most likely caused by an increase in the amount/strength of the hydrogen bonds it is
involved in. This has most likely had effect on Fb and Fc, and has drawn electron density away from these
atoms, resulting in a slight up field shift for both peaks.
A
Fig.5 2D TROSY spectra for P138A-MgF3-G6P (black) and WT (red) overlay (A) and P138L-MgF3-G6P (black) and WT (red)
overlay (B)
The double mutant K145A D10N MgF3-G6P was run 3 times, with a range of G6P concentrations from 0mM
35mM (Fig. 7A). The 2D TROSY spectra overlay of these 3 experiments shows that upon ligand binding there
is no real conformational changes, with only a few chemical shift differences. This suggests that this mutant
may possess an open complex, with a sugar phosphate molecule bound. The fluorine spectra (Fig. 7B) is
interesting as there is no peak for an MgF3 TSA, and this complex is therefore not forming a TSA. Using both
the 2D TROSY and fluorine spectra, it has been hypothesised that there is something different forming upon
ligand binding, and may be a bound bisphosphate. This has previously been observed in the single D10N mutant
active site, and eventually renders the enzyme inactive.
B
Fig. 6 2D TROSY P138A-MgF3-G6P (red) and P138L-MgF3-G6P (black) overlay (A). Fluorine spectra for the P138A-MgF3-G6P (blue)
and P138L-MgF3-G6P (red) overlay (B)
Fig. 7 2D TROSY overlay of K145A D10N apo (black), K145A D10N 17mM G6P, 10mM NaF, 5mM MgCl (red) and K145A D10N
35mM G6P, 10mM NaF, 5mM MgCl (blue) (A). Fluorine spectrum for K145A D10N MgF3-G6P (B)
Future Directions
Perform 1D phosphate NMR to determine what is affecting the structure of the double mutant K145A D10N,
and whether it is in fact a bound bisphosphate. Molecular replacement of the electron density maps of the
crystals, with the known WT structure. This will be analysed together with the NMR data to attempt to
determine the structural changes caused by these mutants. Attempt to crystallise D10N without magnesium
bound, as it has only been previously done in the presence of magnesium. When the D10A plasmid has arrived,
characterise this mutant in the same way as above to determine the differing effects of an asparagine and alanine
mutant of the general base.
Value of the Studentship
Student
The studentship has been a valuable and unique experience, which has enhanced my laboratory experience and
analytical skills. Over 8 weeks, I have had the opportunity to use different scientific methods to determine
structural properties, interpret complex data sets, and work with experts in this field of study. I have gained an
insight into the day-to-day operation of a research lab, which is completely different to any previous laboratory
experience. Before I began this project, I was unsure about a career in research and whether I had the mind-set
to do so, and I believe that without this studentship, I would still not have the self-belief to be strongly
considering completing a PhD in the future.
Supervisor
The studentship has been of great value to our laboratory and Carly has managed to achieve excellent
crystallography data, in some cases to a resolution of 1.06. Through working in a biophysics laboratory, she
has gained a huge variety of skills, from good laboratory practice, to working with new equipment and
interpreting complex data. Her work ethic has helped to maintain the fast pace of the project and it was a
pleasure to have her working in the laboratory.
1.
2.
3.
4.
5.
6.
Zhang, G., Dai, J., Wang, L., Dunaway-Mariano, D., Tremblay, L. W., & Allen, K. N. (2005). Catalytic cycling in
beta-phosphoglucomutase: a kinetic and structural analysis. Biochemistry, 44(27), 94049416.
Baxter, N. J., Bowler, M. W., Alizadeh, T., Cliff, M. J., Hounslow, A. M., Wu, B., Waltho, J. P. (2010). Atomic
details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by
phosphoranes. Proceedings of the National Academy of Sciences of the United States of America, 107(10), 4555
4560.
Dia, J.B, Liu, Y., Ray W.J. Jr, Konno, M. (1992). The crystal structure of muscle phosphoglutamase refined at 2.7
angstrom resolution. Journal of Biological Chemistry, 267(9):6322-6337
Lahiri, S. D., Zhang, G., Dunaway-Mariano, D., & Allen, K. N. (2002). Caught in the Act: The Structure of
Phosphorylated b-Phosphoglucomutase from Lactococcus lactis. Biochemistry, 41(26), 83518359.
Blackburn, G. M. (2003). Comment on The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer
Reaction. Science, 301(5637), 1184c1184.
Griffin, J. L., Bowler, M. W., Baxter, N. J., Leigh, K. N., Dannatt, H. R. W., Hounslow, a. M., Waltho, J. P.
(2012). Near attack conformers dominate -phosphoglucomutase complexes where geometry and charge
distribution reflect those of substrate. Proceedings of the National Academy of Sciences, 109(18), 69106915.
Connor Sampson
University of Kent
Summer 2015
Biochemical Society Summer Studentship Report: The Effects of Varying Translational Accuracy
on Protein Behaviour and Cell Phenotype in S. Cerevisiae.
Introduction
It is well established that while multiple codons may represent a single amino acid, not all codons
are translated with the same degree of accuracy. [1] While this accuracy is affected by a number of
factors, such as relative aatRNA abundance and decoding times, [1] the principle may be used to design
genes which produce proteins of a desired fidelity. Here two versions of GST were created, one using
high accuracy codons (GST_Max) and the other low accuracy codons (GST_Min), in order to study the
effect of these errors in a controlled manner.
Aims
The original aims were to extract both GST_Min and GST_Max from transformed strains of S.
cerevisiae and subject them to mechanochemical analysis to study the functional manifestations of these
errors. During the project, however, difficulties extracting the products in a non-denatured state caused
the studentship, and it's limited timescale, to focus instead upon the in vivo effects of these variants
Experiments
Plasmids containing the desired genes were transformed into a standard BY4741 and a triple
protease mutant (Prot_3; lacking pre1 [Proteasome catalytic subunit] [2], prb1 [Vacuolar protease] [3],
and pep4 [vacuolar protease] [4]) haploid cell lines.
The use of a standard GST affinity column was decided against as it would have acted
selectively, binding only well formed proteins. The use of a N-terminal His-tag was instead used for both
constructs, however this was only accessible under heavily denaturing conditions (8M urea).
As the Mw of GST was known, SDS-PAGEs of purified GSTs were run, the relevant gel slices
excised and destained, and placed into the wells of IEF gels in order to compare charges.
Further to this, new transformants were constructed for strains deficient in particular degradation
pathways (Table 1) in order to study possible in vivo breakdown mechanisms of highly erroneous
proteins, and the associated effects on cell function. Growth curves were constructed for these
transformants, and western blots were carried out to observe the comparative GST levels.
The IEF preparation, involving destaining, renaturing, and the loading of gel slices, was only
partially successful, producing IEF protein "smears". Optimisation is therefore required.
Western blots for BY4741 and Prot_3 colony extracts (Fig. 1) showed greater expression of
GST_Max verses GST_Min in all strains with a PGK1 loading control. (For the purposes of this project,
PGK1 abundance is assumed constant in all mutants.) This was to be expected from codon usage, as
rapidly translated codons are associated with greater accuracy, allowing greater levels to be produced by
the cells. [1] It did not show a clear difference in relative GST abundance between the two strains.
It may be noted that all proteins in Prot_3 showed a decreased molecular weight, although the
exact causes of this are unclear.
The growth curve data showed no significant change in maximum growth rate in the tested
knockout strains for the insertion of either GST_Min or GST_Max. (fig. 2)
Due to time constraints it was not possible to carry out knockout strain western blots in replicate
and as such significant differences cannot be claimed from the data (fig.3), although expression was
confirmed to follow standard patterns, with greater GST_Max than GST_Min abundance in all strains.
Future Directions
Insertion of a penta-Gly linker before the His tag may improve accessibility in the native protein.
Connor Sampson
University of Kent
Summer 2015
The IEF could be optimised through the application of a more controlled loading medium, such as
a homogenised gel slice suspension, or even a solution of extracted protein.
The knockout strain western blots may be carried out in replicate in order to properly examine for
significant differences in strain GST abundance.
Career Aspirations
I previously had difficulty deciding between a career in research and a career in science
communication, fearing that lab work may not be sufficiently engaging. This studentship has done much
to dismiss these fears and I am now significantly more likely to pursue laboratory work.
Value to Myself
The studentship itself represented a highly valuable episode in my academic career, providing
both experience and a greater understanding of laboratory work. I had carried out experiments previously
as part of my degree, however it quickly became apparent that these were very different to actual lab
work where I was now able to pursue a larger project, make decisions based on actual data, and work in a
far more flexible environment. The studentship, while being enjoyable, has provided both the
encouragement and some of the skills to pursue a future career in research.
Value to the lab
"Connor's summer placement provided valuable help and extended an ongoing project, allowing
us to pursue an avenue of exploration which otherwise would have remained unexplored. Connor's
assessment of how properties of DNA sequences which should give rise to identical proteins nevertheless
lead to subtly different behaviour of the expression host enriched ongoing work characterising minor
protein sequence variants of recombinantly expressed proteins. Moreover, Connor road-tested novel
electrophoresis-based procedures which we are now starting to use for the characterisation of
heterogeneity in various gene products. Overall, we value Connor's contribution to the project and the
general life in the lab very highly, and we thank the Biochemical Society for enabling this contribution by
providing Summer Vacation Studentship funding."
Bibliography
[1] D. Tarrant and T. v. d. Haar, Synonymous codons, ribosome speed, and eukaryotic gene, Cellular and Molecular Life Sciences, vol. 71, no.
21, pp. 4195-4206, 2014.

[2] M. C. Morris, P. Kaiser, S. Rudyak, C. Baskerville, M. H. Watson and S. I. Reed, Cks1-dependent proteasome recruitment and activation of
CDC20 transcription in budding yeast, Nature, vol. 423, pp. 1009-1013, 2003.
[3] C. M. MOEHLE, R. TIZARD, S. K. LEMMON, J. SMART and E. W. JONES, Protease B of the Lysosomelike Vacuole of the Yeast
Saccharomyces cerevisiae Is Homologous to the Subtilisin Family of Serine Proteases, MOLECULAR AND CELLULAR BIOLOGY, vol. 7,
no. 12, pp. 4390-4399, 1987.
[4] G. AMMERER, C. P. HUNTER, J. H. ROTHMAN, G. C. SAARI, L. A. VALLS and T. H. STEVENS, PEP4 Gene of Saccharomyces
cerevisiae Encodes Proteinase A Vacuolar Enzyme Required for Processing of Vacuolar Precursors, MOLECULAR AND CELLULAR
BIOLOGY, vol. 6, no. 7, pp. 2490-2499, 1986.
[5] H. H. Lee, Y.-S. Kim, K. H. Kim, I. Heo, S. K. Kim, O. Kim, H. K. Kim, J. Y. Yoon, H. S. Kim, D. J. Kim, S. J. Lee, H. J. Yoon, S. J. Kim,
B. G. Lee, H. K. Song, N. Kim, C.-M. Park and S. W. Suh, Structural and Functional Insights into Dom34, a Key Component of No-Go
mRNA Decay, Molecular Cell, vol. 21, pp. 938-950, 2007.
[6] N. Gallastegui and M. Groll, The 26S proteasome: assembly and function of a destructive machine, Trends in Biochemical Sciences, vol.
35, no. 11, pp. 634-642, 2010.
[7] O. Brandman, J. Stewart-Ornstein, D. Wong, A. Larson, Christopher, C. Williams, G.-W. Li, S. Zhou, D. King, P. S. Shen, J. Weibezahn, J.
G. Dunn, S. Rouskin, T. Inada, A. Frost and J. S. Weissman, A ribosome-bound quality control complex triggers degradation of nascent
peptides and signals translation stress., Cell, vol. 151, no. 5, pp. 1042-1054, 2012.
Connor Sampson
University of Kent
Summer 2015
Tables and Figures:

Knockout Strain
Deficient Protein
Protein function
Phenotype
dom34
Dom34
No Go Decay pathway nonfunctional.
pep4
Saccharopepsin
Endonuclease involved in
ribosome release and mRNA
breakdown in No Go Decay
pathway. [5]
Vacuolar protease, responsible for
activation of multiple zymogens.
[4]
ump1
Proteasome maturation
factor UMP1
Chaperone for proteasome 26S

subunit formation. [6]
Greatly reduced levels of

functional proteasomes.
rqc1
Ribosome quality
control complex
subunit 1
Complex binds stalled ribosomes,

facilitating peptide excision and
ubiquitination. [7]
No degradation of stalled
polypeptides.
Vacuolar protein degradation

heavily compromised.
Table 1: Knockout strains used in the latter experiments.
Maximum Delta
Figure 1. Comparative western blots for BY4741 and delta Prot 3 showing the greater expression
of GST_Max, and the decreased Mw of all proteins from the protease deficient strain. GST_Min and
GST_Max levels vary proportionally in all stains.
0.2
0.15
Control
0.1
0.05
GST_Min
GST_Max
rqc1
ump1
pep4
dom34
Knockout Strain
Figure 2. Maximum exponential growth

rates for the knockout strains, with the
95% confidence interval shown. No
significant difference is seen, indicating
that no version of GST effects maximum
growth rate.
Figure 3. Western blots for the knockout strains in Table 1, showing apparently standard ratios of
GST_Min to GST_Max.
3
Biochemical Society Summer Vacation Studentship Report 2015

Development of a new photoactivable green fluorescent protein for photoactivation
localisation microscopy
Student: Dahlia Eldosoky
Supervisors: Dr Colin Rickman1 and Dr Amy Davies1
1 Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt
University
Background & Aims

The onset of super-resolution microscopy has allowed the detailed description and observation of dynamic processes
in living cells, by surpassing the resolution limit of traditional fluorescence microscopy, which is ca. 250 nm (1).
Targeted labeling of specific cellular components using fluorophores that have the ability to emit at different
wavelengths, allows the examination of cell structures in a live sample. However, the advances in super resolution
microscopy have allowed 3D imaging, observation of cell dynamics and imaging molecular interactions to be possible
(2). One technique in particular uses photoactivatable proteins that allow single protein molecules with distances of a
few nanometers apart - to be resolved by using photoactivation localisation microscopy (PALM) (3). In order for this
technique be successful, the fluorescent proteins photoactivatable behaviour must be exhibited and sufficiently
prominent for there to be detection at the molecular level. PALM at present, is only practically achievable using
photoactivatable proteins that are detected in the red region.
A fluorescent protein from Haeckelia beehleri, called HbGFP1, is a new photoactivatable green fluorescent protein (4)
that has shown fluorescent properties but in spite of this, is not yet optimal for PALM studies due to the long carbonyl
and amino termini.
The aim of the project was therefore to:
Clone and express the truncated versions of HbGFP1
Spectroscopically analyse ensemble switching dynamics

And look at single molecule microscopy of individual purified proteins
Work Carried Out & Results

Clone and express the truncated versions of HbGFP1:
During the 8 weeks, 3 truncated versions of HbGFP1 were made and the proteins of each version were expressed in
bacteria and then purified using several methods. A plasmid DNA backbone with regions encoding StrepTag and
HisTag along with four different primers two used for 5 end and other 2 used for 3) was initially designed prior to
cloning.
Truncation of HbGFP1 at 5, 3 and 3 end of the truncated 5 plasmid (53) were achieved using a Q5 Site-Directed
Mutagenesis Kit. XL10-Gold ultracompetent cells were transformed with the plasmids and bacteria were grown up in
larger quantities, which then the plasmid DNA was purified via miniprep and maxiprep. The sequencing results after
plasmid purification are shown in Figure 1, showing that 39bp were deleted in total 3, 12bp for 5 and 51 bp for 53.
Figure 1. DNA sequencing results of 3 (A), 5 (B) and 53 (C), showing comparison
between truncated versions and original HbGFP1. Gap indicates deletion.
Protein expression was carried out using 5, 3 and 53 plasmids using BL21 ultracompetent cells along with IPTG to
induce protein expression. The proteins have HisTag on 5 end and StrepTag on 3 end, which allowed two forms of
protein purification via StrepTrap and HisTrap column purification using KTAPurifier. Protein fractions that were
eluted from columns were then analysed using SDSPAGE, which the results are shown in Figure 2.
A
25kD
C
Figure 2. 5 StrepTrap gel (A) showed a band of contaminants from Strep
aridin, but this was later removed after HisTrap purification step as shown
on HisTrap gel (A). 3 StrepTrap gel (B) showed smearing, indicating no
bands and hence no protein which was later confirmed by HisTrap gel (B).
53 StrepTrap gel (C) also showed no protein was obtained.
5 purification was successful with protein fractions obtained at under 37kD range but just above 25kD for which
HbGFP1 is 34.6kD. A further purification step was carried for 5 using a spin concentrator and a dialysis cassette but
this also resulted in protein aggregation that formed 2 layers in an eppendorf tube. 3 and 53 protein gels on the
other hand, showed smearing which suggests that the protein aggregated due to lack of stability of protein
conformation.
Overall, 3 truncated versions of purified plasmid DNA of HbGFP were obtained but once protein was expressed,
there was much difficulty in purifying the proteins. In terms of future directions, 3 and 53 truncations are evidently
not possible and so further optimisation for example buffer composition, pH and temperature control - for
purification of 5 truncation is required in order for spectroscopic analysis to be carried out, and, to ultimately look at
single molecule microscopy of individual purified proteins.
Value of Studentship
From student:
The studentship has given me an opportunity to spend time in a research laboratory, allowing me to gain an insight
of both the theoretical and practical aspect of a research environment. I was able to master the techniques of cloning
and protein expression, both of which I was unfamiliar with - in a practical sense - at the start of the project. As a
result, it has extended my practical ability, along with gaining the confidence of being able to apply my academic
theory in something I had no experience in. Ultimately, the whole experience was very valuable; it has boosted my
confidence for when I come to tackle my Masters project and has confirmed my interest in hopefully obtaining a PhD
in the future. I would like to thank the Biochemical Society for giving me this chance to broaden my scope and to the
lab for their constant support and assistance throughout the 8 weeks.
From supervisor:
Dahlia has been a great help over the 8 week summer project. She has made excellent progress and quickly learned
the new skills required to perfrom this project. This project is being continued by Amy Davies (who supervised
Dahlia on a daily basis) and any resulting publication will include some of the work performed by Dahlia this
summer.
References
(1) J. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, 2006.

(2) M. Fernndez-Surez, A. Y. Ting, Nat. Rev. Molecular Biology, 9, 931-932 (2008)
(3) E. Betzig, G.H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, Science, 313, 1643 (2006)
(4) S. H. D. Haddock, N. Mastroianni, L. M. Christianson, Proc. R. Soc., 277, 1155 (2010)*
* After completion of this project the initial article was retracted. However, this was merely due to incorrect
identification of the host species and the DNA sequence, protein sequence, spectroscopic properties and all other
findings in the original publication and in this project remain valid.
CONSTRUCTION OF BILE-ACID REPORTER IN BACTERIAL COMMENSAL STRAIN

Daniel de la Torre - University College London - Computational Systems and Synthetic Biology Lab (Prof. Geraint Thomas)
Fluorescence analysis
BACKGROUND AND AIMS
Ongoing studies like the Human Microbiome Project and
MetaHIT are slowly unravelling the physiological importance
of the symbiotic relationship between humans and microbes
and providing us with new insights into the role of
1,2
microbiota in human health and disease . The commensal
nature of the microbiota and its constituents provide a
number of well-tolerated microorganisms that could
potentially exploit the intestines as an entry point and act as
vectors for therapeutically relevant synthetic genetic
circuits. An example of this is the use of commensal E. coli
3
strain NISSLE1917 for targeting Vibriocholerae infection ,
4
5
diabetes and obesity .
The primary and secondary bile acid axis in the intestines is
6
thought to be implicated in microbial pathogenesis ,
7
8
degenerative diseases and gastric cancers . Hence, there is
a growing need for tools to monitor and investigate the
complex pathways and networks involved in bile acid
metabolism. Recently, the bbr_0838 gene in Bifidobacterium
breve was found to encode a bile-inducible membrane
protein which provides the bacteria with tolerance against
9
bile acids . The promoter region of bbr_0838 was shown to
activate in the presence of bile acids using a gusA reporter
assay. The aim of this project was to investigate whether
this promoter region could be used for the creation of a new
investigative tool for monitoring the bile acids axis in the
intestines, by cloning into a bacterial fluorescent reporter
plasmid and transformation into commensal strain
NISSLE1917. In addition, construction of a simple model for
this construct could help determine whether this reporter
plasmid could be used to monitor physiological levels of bile
acids in vivo.
DESCRIPTION OF WORK
Construction of bile-acid reporter strain

The BBr_0838 promoter region was synthesised with
appropriate restriction sites on either end, for cloning into
the high-copy OG241 plasmid from Oxford Genetics,
upstream of a GFP. The size of the fragment was 304 bp.
Digestion with adequate restriction enzymes was carried out
in both the insert and plasmid, followed by gel-extraction
and DNA purification, for posterior ligation of the BBr_0838
promoter into the OG241 backbone, resulting in the
BBr0838-OG241 reporter plasmid. This plasmid was
transformed in E. coli DH5, and colonies were picked and
grown overnight for further plasmid purification. The
BBr0838-OG241 reporter plasmid was then transformed into
E. coli NISSLE 1917, and its identity confirmed by Sanger
sequencing (service was provided by Source BioScience).
In order to analyse the inducibility of the system, DH5 cells

bearing the BBr0838-OG241 plasmid were grown in 1ml LB
medium shaking at 37 C. Plasmid-free DH5 and DH5 cells
carrying insert-free OG241 were used as a negative controls.
When mid-exponential phase was reached, cultures were
induced with either 50M deoxycholic acid or 0.025%
cholate. They were then incubated for another 7 hours
shaking at 37 C. 200 L of each culture were then
centrifuged at 10000rpm for 2 minutes, and the supernatant
discarded. The cells were then resuspended in 400 L PBS.
The fluorescence intensity of the samples was measured in
TM
an Accuri C6 flow cytometer. The fluorescence data from
this assay was processed using the R programming language.
RESULTS AND DISCUSSION
Construction of both DH5 and NISSLE1917 bile-acid
reporting strains was successful, as was verified by Sanger
sequencing. However, the inducibility assay yielded
unexpected results. While in the presence of deoxycholic
acid and cholate the construct shows strong expression, in
the order of 10-fold, this is also the case in the absence of
bile acids. The construct presented constitutive expression
and hence lacked the bile-acid sensing capacity (Figure 1).
Previous characterisation of this promoter by Ruiz et al. used
a similar genetic construct, but employed B. breve (the
species that originally hosts this promoter in its genome) as
a chassis instead of E. coli. The constitutive nature of the
promoter led us to think that our system might be missing a
repressor which was present in the native strain.
Bioinformatic analysis of the region upstream of the
promoter revealed the presence of a hypothetical protein
BBr_0834 (ABE95519.1), which has not been characterised
but is predicted to contain an AAA+ ATPase domain. This
type of protein has been previously shown to be involved in
switching between different states of gene expression in
10
yeast . BBr_0834 could potentially be involved in restructuring of the chromatin, with a resulting halt in
expression, or in inhibition of another activator. The precise
mechanisms of regulation by the BBr_0838 promoter have
not yet been fully characterised, and further research would
be required for determining whether this protein effectively
plays a role in bile acid induction and how bile acids would
contribute to relieving the repression. A simple ODE model
was constructed in Matlab to confirm that the presence of a
repressor would indeed make the system inducible.
DEVIATIONS FROM ORIGINAL PROPOSAL
The unexpected results with regards to the bile acid sensor,
and the unfeasibility of troubleshooting the system in terms
of available time due to the wider scope of this new project,
drove me to start working on a different acid-sensor which
was of interest to the group. This sensor, constituted by the
VALUE OF THE STUDENTSHIP

To the student:
This has been a great opportunity for me to get more
familiar not only with molecular biology techniques, but also
with the design and planning of a project from beginning to
end. It was also confirmation that in science things dont
always go as expected, and that the paths towards testing
and confirming a hypothesis are often more intricate and
tortuous than they might seem in the first place. But
perhaps most importantly, and although this is not
extensively reflected in the results, it has introduced me to
the field of computational modelling and systems biology,
which seem like invaluable tools for making biology more
reliable, predictable and reproducible, all mandatory
requisites in the construction of new synthetic genetic
devices and systems. After this experience it is clearer to me
that I would like to pursue a career in this area, and play an
active part in the synthetic biology era.
cadBA promoter region, is induced in conditions of acidic
11
pH, and its strength is enhanced by anaerobiosis . The
Figure 1. Constitutive GFP expression in the BBr0838OG241 construct. For both 0.025% cholate (A) and 50
uM deoxycholate (B), the top plot represents the nopromoter control, and the bottom plot represents the
construct including the BBr0838 promoter. The plots
were obtained using the R programming language; the
code extracts medians for the values and then
calculates means and standard deviations, fits the
parameters to a Hill function and then plots the density
on the y axis against the fluorescence on the x axis.
fragment was amplified by PCR from genomic DNA, and

cloned into OG241 using the techniques described above.
DH5 cells were grown in LB and diluted in fresh minimal
medium, previously adjusted to pH 5.8 or pH 7.6 and
appropriately buffered. Unfortunately, fluorescence analysis
did not show induction of gene expression in acid stress
conditions and hence this reporter requires further
troubleshooting.
FUTURE DIRECTIONS
The precise molecular mechanisms of bile-acid induction of
the BBr_0838 promoter remain to be elucidated, and the
potential effect of hypothetical repressor BBr_0834 on gene
expression could constitute a starting point for this
investigation. If the inclusion of this protein in the system
resulted in a functional bile acid inducible reporter
construct, more data would need to be gathered towards
estimation of the crucial parameter values for the
mathematical model. Ultimately, the bile-acid sensing
NISSLE1917 strain could be employed in in-vivo studies of
bile-acid reporting in mice, as a step towards its
implementation in humans for diagnostic and therapeutic
purposes.
To the supervisor:
Daniel has done very useful work in his initial
characterisation of a potential bile acid reporter construct
for eventual use in in vivo experiments. While it is
unfortunate that the reporter system didnt work first time,
Daniel's excellent and meticulous work has given us useful
additional insights into the complexities in the problem and
suggested potential avenues for further investigation and
possible solutions. In addition Daniel has helped to further
establish our techniques and procedures for working
successfully in commensal strains of E.coli suitable for our
future investigations.
REFERENCES
1. Consortium, T. H. M. P. Structure, function and diversity of the
healthy human microbiome. Nature 486, 207214 (2012).
2. Qin, J. et al. A human gut microbial gene catalogue established by
metagenomic sequencing. Nature 464, 5965 (2010).
3. Duan, F. & March, J. C. Engineered bacterial communication
prevents Vibrio cholerae virulence in an infant mouse model. Proc.
Natl. Acad. Sci. 107, 1126011264 (2010).
4. Duan, F., Curtis, K. L. & March, J. C. Secretion of Insulinotropic
Proteins by Commensal Bacteria: Rewiring the Gut To Treat
Diabetes. Appl. Environ. Microbiol. 74, 74377438 (2008).
5. Chen, Z. et al. Incorporation of therapeutically modified bacteria
into gut microbiota inhibits obesity. J. Clin. Invest. (2014).
doi:10.1172/JCI72517
6. Buffie, C. G. et al. Precision microbiome reconstitution restores bile
acid mediated resistance to Clostridium difficile. Nature 517, 205208
(2015).7. Jones, M. L., Martoni, C. J., Ganopolsky, J. G., Labb, A. &
Prakash, S. The human microbiome and bile acid metabolism:
dysbiosis, dysmetabolism, disease and intervention. Expert Opin.
Biol. Ther. 14, 467482 (2014).
8. Bernstein, H., Bernstein, C., Payne, C. M., Dvorakova, K. &
Garewal, H. Bile acids as carcinogens in human gastrointestinal
cancers. Mutat. Res. 589, 4765 (2005).
9. Ruiz, L. et al. A bile-inducible membrane protein mediates
bifidobacterial bile resistance. Microb. Biotechnol. 5, 523535 (2012)
10. Wilcox, A. J., Laney, J. D. A ubiquitin-selective AAA-ATPase
mediates transcriptional switching by remodelling a repressorpromoter DNA complex. Nat. Cell. Biol. 11, 1481-86 (2009).
11. Cper, C., Jung, K. CadC-mediated activation of the cadBA

promoter in Escherichia coli. Journal of Molecular Microbiology and
Biotechnology 10, 26-39 (2006)
Developing a method to detect cysteine oxidation of the glucose-sensor enzyme

glucokinase
Name: Daniel O. Fajonyomi.
Supervisor: Dr Catherine Arden, Institute of Cellular Medicine, Newcastle University.
Gl ucokinase (GK) catalyses the first step of glucose metabolism, i ts phosphorylation to glucose 6-phosphate, a nd
s erves as the glucose sensor for gl ucose-stimulated insulin s ecretion i n tissues with a major role i n blood glucose
homeostasis i ncluding the pancreatic beta-cells. The activity, s tability a nd/or expression of GK i s regulated by a number of
pos t-translational modifications i ncluding oxidation of its cys teine residues which i nhibits enzyme a ctivity. Susceptibility of
GK to oxi dation i s a predisposing factor to the instability of several GK-MODY mutants that ca use diabetes i n man a nd
endogenous GK i s inactivated by elevated oxidative s tress, this supports a model whereby endogenous GK a ctivity i s
i nhibited by thi ol oxidation i n conditions of a nti-oxidant depletion and/or reactive oxygen s pecies production which is
pa rti cularly relevant gi ven that i ncreased oxidative stress is assumed to be a critical component in the impairment of beta cel l function during the development of type 2 diabetes. Whether modulation of GK a ctivi ty/stability by changes i n its thiol
s ta tus plays a role in the development of type 2 di abetes remains to be determined.
The aim of this project:
The a im of this research project was to develop the Purification of Reversibly Oxi dised Proteins ( PROP) method
to detect oxidation of glucokinase (GK) i n pancreatic beta-cell extracts, in order to a chieve this the research project
i nvol ved three s ets of experiments which are: I) Optimisation of the PROP technique with GK IP using recombinant proteins
II) Application of the PROP technique to beta-cell extracts treated with oxidising a gents III) Application of the PROP
technique to beta-cells exposed to i ncreased oxidative s tress.
A description of the work carried out (Methods & Results):
The PROP method involves four major steps outlined below:
1.
2.
3.
4.
Sa mples treated with, and without, alloxan (which oxidises cys teine residues on glucokinase) are denatured a nd
trea ted with NEM which irreversibly blocks reduced thiols on gl ucokinase.
NEM i s removed by protein precipitation and DTT is then used to reduce the thiols oxidised by a lloxan.
DTT i s removed and the newly-reduced thiols are then tagged with biotin.
Tota l Glucokinase protein is i mmunoprecipitated using a GK a ntibody a nd the unbound and boun d (IP) fractions
a re resolved using western blotting.
Fi gure 1: Outl ine of PROP method.
Fi gure 2: Outl ine of GK i mmunoprecipitation technique.

Typi ca lly two gels are prepared for SDS-PAGE resolution of both the control and alloxan samples, and both samples
a re ra n on each gel i n order to obtain two separate PVDF blots. One blot i s blocked in 5% mi lk and then i ncubated with a
pri ma ry a ntibody a nd then Rabbit-HRP (secondary a ntibody) to determine the total a mount of glucokinase in each s ample,
whi le the other blot is blocked i n 1% BSA and then i ncubated with Streptavidin-HRP which binds to biotin a llowing
vi s ualisation of the level of oxidation in both samples.
Opti misation of the PROP technique with GK IP using recombinant proteins: In these experiments recombinant GST tagged
gl ucokinase a nd cl eaved glucokinase (GK without the GST-tag), both of which are isolated from E.coli, were used as the
gl ucokinase sources. In the first e xperiment recombinant GST-GK protein samples were treated with, a nd without (con trol
s a mple), alloxan a nd the PROP method (without immunoprecipitation) was used to detect oxi dation. Using this a pproach, I
wa s able to demonstrate that treatment of GST-GK wi th alloxan caused oxidation of glucokinase a s evident from an
i ncrease in the HRP-streptavidin signal, i ndicating an i ncrease in biotin-tagging a nd thus oxidation.
I then repeated this experiment using recombinant glucokinase protein from which the GST-tag had been
removed (cleaved GK) a nd achieved similar results which confirmed that the increase i n the Strep-HRP signal from previous
experiments was due to the oxidation of glucokinase a nd not the GST-tag.
Si nce the aim of the project is to develop a method to detect the oxidation of glucokinase within cell l ysates, i t is
es sential to isolate GK from other unwanted proteins using immunoprecipitation. We therefore confirmed that the IP
protocol did not alter the biotin-tagging by i ncorporating GK i mmunoprecipitation into the protocol using recombinant GK
s ources. I was able to s how that total GK could be i mmunoprecipitated s uccessfully wi th the current protocol and produce
res ults similar to those obtained without the IP protocol.
Appl ication of the PROP technique to beta-cell extracts treated with oxidising agents: The second s et of experiments aimed
to s uccessfully a pply the PROP method to the detection of GK oxidation in cell extracts from INS1E beta -cell lines treated
wi th a lloxan, two experiments were done towards this aim. First we confirmed that we could IP GK from cell l ysates by
ca rryi ng out the IP protocol as described a bove using cell extracts a nd were able to s how that total GK ca n be
i mmunoprecipitated from beta-cell extra cts s uccessfully.
After s uccessfully immunoprecipitating total GK from cell extracts, we determined whether we could detect
oxi dation of gl ucokinase from cell lysates using PROP followed by IP. However, when PROP (wi th alloxan oxidation) and IP
were i ncluded i n the protocol, the IP and unbound fractions could n ot be vi sualised on the total GK a nd Streptavidin-HRP
bl ots. It is believed that this was due to a loss of protein during the protein precipitation s teps in the PROP method a nd
therefore optimisation of the protein precipitation steps would be required to achieve a s uccessful experiment.
Deviations from original proposal:
Due to ti me constraints it was not possible to begin the last phase of experiments (Application of the PROP
technique to beta-cells exposed to i ncreased oxidative s tress).
Future directions in which the project can be taken:
As i ndicated previously, there is a need to optimise protein precipitation to ensure that sufficient protein is
reta i ned to a llow for i mmunoprecipitation. Once the technique is a ble to detect glucokinase oxida tion i n this a rtificial
s ys tem, the technique ca n then be used to determine whether glucokinase is oxidised under conditions related to type 2
di a betes which will i nclude i ncreased oxidative s tress a nd a lso nutrient excess (glucolipotoxicity). This will eventually l ead
to the a pplication of the technique to detect glucokinase oxidation i n animal models of diabetes a nd potentially human
ti s sue from type 2 di abetic patients.
Value of the studentship to the student:
Thi s research placement with Dr Arden a nd h er team have truly been i nvaluable, during this 8-week period I feel I
ha ve not only gained insight a nd a ppreciation for research but greater interest in the work being done in diabetes research
through seminars and presentations I was gi ven the opportunity to a ttend a nd i nteractions with other researchers in the
l a b. In addition I have also had the opportunity to l earn new lab techniques, use techniques I was a lready familiar to new
probl ems a nd gain l ab skills which I believe would be i ncredibly useful during my fi nal year lab-based research project. All
of thi s was made possible by Dr Arden a nd her team and the grant provided by the biochemical society.
Value of the studentship to the lab:
It wa s a pleasure to have Daniel in the lab. He quickly a djusted to the laboratory environment and interacted well
wi th s tudents and research staff. During his time, he gained experience in techniques s uch as i mmunoprecipitation, cell
l ys is a nd western blotting. The data he has generated will be used for future grant applications to expand on this project.
UNIVERSITY OF ST ANDREWS
BIOMEDICAL SCIENCES RESEARCH COMPLEX
BIOCHEMICAL STUDENTSHIP 2015
ELYSE FISCHER
CASPOSON CAS1 PROBING THE BASIS FOR CRISPR ADAPTIVE IMMUNITY

Supervisor: Professor Malcolm White
Daily Supervision: Shirley Graham and Clare Rollie
Figure 1: Cas1 homodimeric

structure showing canonical
nuclease active site
Background
CRISPR-Cas is a prokaryotic adaptive immune system which provides protection against invading genetic
elements through homology-directed detection and destruction. The CRISPR loci comprises of a leader sequence
followed by an array of short palindromic repeats interspersed by small spacer sequences. As well as a variety of
CRISPR associated (Cas) genes. Spacers, captured from viruses, are integrated in the host genome and provide a
memory of past infections. Upon reinfection, the CRISPR locus is transcribed generating pre-CRISPR RNA (precrRNA). This transcript, subsequently processed and loaded into Interference or Effector ribonucleoprotein
complexes, utilizes the crRNA to detect and degrade previously encountered foreign entities (1).
This method of capturing foreign DNA and integrating it into the host genome is known as Adaptation, and
requires Cas1 and Cas2 (2). Cas1, a homodimer, is believed to act as a metal-dependent nuclease (figure 1), cleaving the
CRISPR locus specifically at the integration site to allow spacer addition (3). Cas1 is reported to preferentially cleave
branched DNA substrates, particularly ones which model replication forks (4). Recently, a group of Cas1 orthologues
called Casposons has been identified from a family of transposons (5). These Casposons are believe to play a role in
transposon integration and excision and may underlie the evolution of the CRISPR system.
Project Aims
The project aimed to express and purify a Casposon-derived Cas1 using a commercial synthetic gene. Furthermore,
to determine specificity and reaction mechanism of the Casposon Cas1 against model DNA substrates through
biochemical assays.
Experimental Procedure and Results
Cloning and Expression of Casposon Cas1
Initial cloning experiments and
subsequent troubleshooting failed to
insert the MmaCas1 synthetic gene into
a standard pHisTEV expression vector.
Five new vectors were trialled and
successful ligation was achieved in
three separate vectors.
All three clones were transformed
into C43 expression cells, and induce
with Isopropyl -D-1-thiogalactopyranoside (IPTG). Optimal
temperature and growth time for
Casposon Cas1 expression was
determined using mini expression trials
in a BioSprint machine and analyzed
via SDS-page (Figure 2A). Two
expression conditions were chosen with
promising protein solubility, Cas1pET17b induced at 16C overnight, and
Cas1-pHisTEV induced at 37C for 4
hours. However, expression did not
follow mini trials when large sale
Figure 2: Top Left: Polyacrylamide gel showing Cas1-pET17b mini expression trials. IPTG induction at
16C, 25C, 37C and at 4hours and overnight. Top right: HisTrap Column FF chromatograph of Cas1cultures were induced and a large
pET17b induced IPTG at 16C overnight. Bottom left: Subsequent S200 gel filtration chromatograph.
percentage of the total protein was
right: SDS-page analysis of Cas1-pET17b sample post gel filtration. First well following the
insoluble. Despite this, purification was Bottom
protein ladder contains pre-gel filtration sample, remaining wells contain fractions taken from poststill continued.
gel filtration.
Purification of Casposon Cas1

Protein purification of Cas1-pET17b was completed using a TEV-protease cleavable polyhistidine tag on the Cterminus of the protein. Supernatant prepped in Tris was passed through a HisTrap FF column for metal affinity
chromatography using an AKTA purification system and eluted using a high concentration of competitive imidazole
(Figure 2B). The polyhistidine was then cleaved off using TEV protease and a second nickel column was run. A final
polishing step of size exclusion chromatography using gel filtration was completed (Figure2C). SDS-page was used to
analyze fractions from all steps (Figure2D).
Several complications, including Cas1 precipitation after TEV cleavage and concentrating, resulted in only 400ul at
.535mg/ml of pure protein being obtained. A second round of Casposon Cas1 purification was completed. Two different
vectors were tested under different expression and purification conditions in an effort to reduce the amount of protein
precipitation and aggregation. Most notably, the polyhistidine tag was not cleaved by TEV protease and purification
buffers with a lower pH of 6.5 were used. Despite the changes made, final results did not fare much better. Nevertheless
plenty of protein was obtained in the three batches to test Casposon Cas1 activity in biochemical assays.
Substrate Binding and Cleavage Assays
Four DNA oligonucleotide substrates were designed
(Figure4) to test the ability of Casposon Cas1 to catalyse transesterification on branched DNA substrates. Polyacrylamide
electro-mobility shift assays (EMSA) were used for analysis. The
four substrates included ssDNA, dsDNA, ssDNA flap, and a 3Figure 3: Hybridized DNA oligonucleotide substrates used to
way-junction. Substrates were assembled from oligonucleotides by test Casposon Cas1 activity. From left to right: ssDNA,
dsDNA, ssDNA flap, 3-way junction. The X50 blue strand was
hybridization and gel purification, and fluorescently labelled with
fluorescently labelled with FAM (yellow dot).
6-flourescein amidite (FAM) for visualization.
Casposon Cas1 was found to strongly bind all four
substrates, with the strongest preference for the ssDNA and ssDNA flap substrates. This corresponds with results seen for
the E.coli Cas1 previously tested in the lab. However, cleavage assays showed no activity for Casposon Cas1 in any of the
substrates, compared to the E.coli Cas1 control, which showed non-specific cleavage of ssDNA, and specific cleavage of
the 5-ssDNA flap substrate at the branch site.
Future Directions
Future work is needed to acquire more pure protein, especially in order to achieve enough for crystallization trials.
This would include optimizing expression conditions to obtain better solubility and refining purification conditions to
limit the amount of protein precipitation which occurs. Furthermore, much of the mechanism and activity of Casposon
Cas1 still eludes us, therefore further biochemical assays could be completed on a larger variety of substrates.
Casposon Cas1 was not found to contain any cleavage activity in my project. I plan to return later in the semester to
complete a trypsin digest of Casposon Cas1. The hope is that this will clarify whether the protein was properly folded
despite containing an extra helix-turn-helix domain, and possibly allow removal of the extra domain, in an effort to
increase its cleavage activity.
Departures from Original Proposal
Due to time constraints caused by complications in cloning and purification further biochemical activity assays
were not completed. Only four of 10-12 originally designed substrates were tested.
My lab experience this summer was absolutely phenomenal in all aspects. By my own choice, I prolonged my
project for an extra 4 weeks because I did not want to stop. It cemented my resolve to pursue a PhD following my
undergraduate studies and increased my confidence and perseverance as a scientist.
I learned and refined invaluable experimental techniques including cell transformation, protein expression and
purification, DNA substrate construction, and fluorescent imaging. All which will be extremely helpful in preparing me
for not only my graduate studies but also my dissertation research starting this October, completing structure studies on
the MscL mechanosensitive channel with Professor James Naismith.
My experience was very cooperative but also exceedingly independent. In some ways I am glad many steps of my
project did not work on first try. Obtaining disappointing results not only forced me to troubleshoot and gain more
experience redoing steps, but also taught me to never be disheartened when things dont go how you plan.
My work was also highly beneficial to my lab. Cas1 Casposon had never been worked on before so my project
built a foundation of knowledge to which others may use to carry on further work in the area.
References
1) Reeks, et al. (2013) Biochem. J., 453, 155-166.
2) Makarova, et al. (2006) Biol Direct, 1, 7.
3) Wiedenheft, et al. (2009) Structure, 17, 904-912.
4) Babu, et al. (2011) Mol. Microbiol., 79, 484-502.

5) Krupovic et al. (2014) BMC Biol. 12:36
A Role of SH3BP1 in Spindle Assembly

Frankie Butera
My placement took place in Dr Chris Bakals Dynamic Cell Systems laboratory at the Institute of Cancer
Research (ICR), under the direct supervision of Dr Alexis Barr. Over eight weeks I investigated the assembly
of bipolar spindles, which are critical for the correct segregation of chromosomes during mitosis. chTOG is a
gene with a major role in spindle pole organisation and microtubule stabilisation (Gergely et al., 2003).
Depletion of chTOG results in a multipolar spindle phenotype in a number of cell lines, including HeLa cells
(Barr & Bakal, 2015). In an image-based double RNAi screen of a RhoGEF/GAP library, the gene SH3BP1 was
identified to genetically interact with chTOG (unpublished data). SH3BP1 has GAP activity and employs this
to regulate a number of cellular processes, including activation of Cdc42 in junction assembly (Elbediwy et
al., 2012) and inactivation of Rac1 at the front of cells to drive motility (Parrini et al., 2011). This project
aimed to validate the screen result using independent siRNAs and ultimately determine if SH3BP1 has a
role in spindle assembly. My original proposal had involved looking at p21 dynamics during the G1/S
transition in human cells, however, this had already been achieved before I began my placement.
Procedures and Results

Throughout the studentship, I maintained two HeLa cell lines at 37C in DMEM. One cell line had chTOG
shRNA that was created using a Doxycycline-inducible shRNA system, whereby addition of Doxycycline
stimulates the expression of chTOG shRNA and RFP mRNA. RFP expression was later used to select cells
expressing the shRNA. The other cell line was created using the same system but with non-silencing (NS)
shRNA.
I investigated whether co-depletion of chTOG and
SH3BP1 has an effect on spindle morphology by carrying
out a reverse transfection of the two cell lines with SH3BP1
siRNA. As a negative control, non-targeting siRNA was
used. Co-depletion of chTOG and TUBG1 is known to
rescue a bipolar phenotype, and so TUBG1 siRNA was used
as a positive control to verify that the reverse transfection
worked. Four different SH3BP1 siRNAs were used
independently to ensure that the phenotype observed was
not due to off-target effects, and a pool of these four
SH3BP1 siRNAs was also used. Prior to reverse transfection,
I carried out a real-time PCR to check that the siRNAs
effectively deplete the SH3BP1 mRNA. Figure 1 shows that
the siRNAs have varying efficiencies, with the SH3BP1 pool,
Figure 1: fold-changes (2-Ct ) from quantitative
SH3BP1 10 and SH3BP1 12 having significant reduction in
PCR, normalised to a housekeeping gene (GAPDH)
and adjusted to the control cDNA (TOG con) set to
SH3BP1 mRNA.
1.0.
For the reverse transfection, the control and SH3BP1
siRNAs were plated onto a 384-well plate and Lipofectamine RNAiMAX (a transfection reagent), 2000 cells,
and Doxycycline were added to each well. Prior to fixing, MG132 was added to each well in order to arrest
the cells in metaphase. The cells were fixed with formaldehyde and permeabilised with TritonX-100. I
stained with rat anti-alpha-tubulin to visualise the mitotic spindles and rabbit anti-PHH3 to help mark
mitotic cells, as serine-10 and serine-28 of histone H3 is only phosphorylated during mitosis. I also stained
with Hoescht (DNA stain) to select and segment all nuclei. Images were then taken of each well using the
Perkin Elmer Opera high-throughput confocal microscope, and the images were uploaded to Columbus, an
image analysis system for automated phenotypic screening. I customised and ran a script on Columbus to
select the mitotic nuclei based on cell roundness, tubulin intensity, and PHH3 intensity. The script also
removed any nuclei on the border of an image in case these had extra poles outside of the image. The final
steps of the script counted the number of spindle poles in each cell using a spot finder function, and
classified the spindles with more than two poles as multipolar.
Figure 2: reverse transfection of HeLa cells results in a rescue of bipolar spindles in cells. (a) cells with typical bipolar spindles, (b)
cells with typical multipolar spindles (images from Perkin Elmer Opera with tubulin staining visualised). (c) mean % cells with
multipolar spindles, calculated from six technical repeats for each experiment, for each shRNA and siRNA combination (calculated
using Columbus software). Each square represents one biological repeat.
This experiment was repeated twice and the results from image analysis are shown in Figure 2c. When
chTOG is depleted, the percentage of cells with multipolar spindles increases. When comparing the control
siRNA treatment and the SH3BP1 siRNA treatments, there is a reduction in the percentage of multipolar
spindles when chTOG and SH3BP1 are co-depleted, thus rescuing a bipolar spindle phenotype. This
suggests that SH3BP1 has a genetic interaction with chTOG and plays a role in spindle assembly.
Future of this project

Depletion of SH3BP1 protein by siRNA will be checked using a Western blot. Furthermore, we will verify
that SH3BP1 depletion is specifically causing this rescue of bipolar spindles in chTOG-depleted cells by cotransfecting the SH3BP1 cDNA that is resistant to siRNA into these depleted cells and seeing if this prevents
the rescue in phenotype. We can also check if the GAP activity of SH3BP1 is relevant by adding SH3BP1
cDNA with defect GAP activity.

This studentship has enabled me to explore biology from a perspective beyond my undergraduate degree.
I have been able to gain an honest experience of laboratory research and, as a result, am more prepared
and determined for a career in cell biology research. Moreover, I have learnt many protocols key for this
kind of research, including transfection, quantitative PCR and Western blot, and I now understand how to
use these experiments to test original hypotheses about cellular mechanisms. Finally, during my time at the
ICR I have discovered areas of cancer biology that I may decide to specialise within in the future, such as
cancer and metabolism, and this will be invaluable when applying for a PhD project.
References
Ba rr, A. R. & Ba kal, C. (2015) A s ensitised RNAi s creen reveals a ch -TOG genetic interaction network required for spindle assembly.
Scientific Reports. 5.
El bediwy, A., Zi hni, C., Terry, S. J., Cl a rk, P., Ma tter, K. & Ba l da, M. S. (2012) Epi thelial junction formation requires con finement of
Cdc42 a cti vity by a novel SH3BP1 complex. The Journal of Cell Biology. 198 (4), 677-693.
Gergely, F., Dra viam, V. M. & Ra ff, J. W. (2003) The ch -TOG/XMAP215 protein is essential for s pindle pole organization in human
s omatic cells. Genes & Development. 17 (3), 336-341.
Pa rri ni, M. C., Sa dou-Dubourgnoux, A., Aoki, K., Kuni da, K., Bi ondini, M., Ha tzoglou, A., Poullet, P., Formstecher, E., Yeaman, C. &
Ma ts uda, M. (2011) SH3BP1, a n exocyst-associated RhoGAP, inactivates Rac1 a t the front to drive cell motility. Molecular Cell. 42
(5), 650-661.
Investigating plant calcium response in plant-aphid

interactions
Student: James Canham
Supervisor: Dr Tony Miller
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH. UK.
Background and Aims

The phloem-feeding green peach aphid, Myzus persicae, is a prolific pest to a number of agriculturally
important crops and is responsible for substantial crop losses globally. The plant-aphid interaction remains
1
poorly understood and the reasons behind the success of M. persicae are not fully elucidated . During an
incompatible interaction between plant and aphid, pattern recognition receptors (PRRs) specifically bind
2
herbivore-associated molecular patterns (HAMPs) triggering cell signalling and plant defence responses . The
2+
early dynamics of this signal is unknown but Ca and reactive oxygen species (ROS) bursts have been
implicated. M. persicae can supress plant signalling, overcoming the plant defence response using effector
3
molecules such as Mp10 .
2+
Evidence for a central role of a Ca -interacting kinase, CIPK3.2 in the plant-aphid interaction was identified
during RNA-seq experiments. This led to the requirement for CIPK3 knock out (KO) mutants in Arabidopsis for
further investigation. An aim of this project was determine whether the CIPK3 KO phenotype is Arabidopsis
ecotype specific by replicating published results in the Ws mutant (cipk3-1) and screening new CIPK3 T-DNA
insertion lines in the Col background. M. persicae feeds and successfully reproduces on Col-0 and Ws
Arabidopsis ecotypes.
2+
To measure the plant response during an aphid interaction, a Ca reporter, GCaMP6 has been genetically
engineered in A. thaliana plants. The potential role of the vacuolar two pore channel 1 (TPC1) was investigated
2+
and the aphid effector Mp10 was knocked-down using RNAi technology to discover its effect on Ca bursts
during the interaction.
Work carried out

Extraction and purification of plant and aphid DNA and RNA was carried out in order to perform analytical PCR
and RT-PCR techniques. Germination assays were performed to test for cipk3-1 KO osmotic stress germination
phenotype using 150 mM NaCl strength MS media and stereo microscopy to screen the seedlings.
2+
Fluorescence stereo microscopy was used to image Ca real-time in GCaMP6 reporter plants during the
Arabidopsis-M. persicae interaction. Image frames were analysed for their fluorescence intensity using ImageJ
and data was investigated using the statistical package, GenStat. Col-0 and TPC1 KO plants (tpc1-2) were
2+
imaged and compared to assess the plant dependence on the vacuolar Ca amplifier TPC1 during the
interaction. Aphid populations were cultured on dsMp10 plants in order to knockdown the putative effector,
Mp10, using RNAi technology. The aphids were tested for the knocked-down effector by qPCR. The Mp10
2+
knockdown aphids were exposed to Col-0 reporter plants to test the effect of Mp10 on the Ca burst during
the interaction.
Results and discussion

CIPK3 KO osmotic stress germination phenotype is Arabidopsis ecotype dependent
Successful replication of the CIPK3 osmotic stress germination phenotype was achieved in the Ws background,
but CIPK3 KO lines in the Col background failed to exhibit a similar phenotype
4
(Fig. 1) .
Figure 1. Germination of CIPK3 KO seed is
sensitive to osmotic stress conditions in the Ws
background but not in the Col background.
These data show that the Arabidopsis CIPK3 KO germination phenotype is significantly reduced in 150mM
NaCl relative to Ws-0, but the same osmotic stress phenotype was not replicated in the Col-0 background.
2+
M. persicae induces a localised but not systemic Ca

interaction which is dependent on TPC1
burst in Arabidopsis during a plant-aphid
2+
GCaM6 Ca reporter plants were imaged during an aphid interaction and frames were analysed for changes in
fluorescence intensity using ImageJ (Fig. 2).
Figure 2. Fluorescence intensity within a specified
region of interest (ROI) of imaged GCaMP6 Col-0
and tpc1-2 reporter plants. (A) Col-0 aphid vs. no
aphid samples at a systemic ROI. (B) Col-0 aphid
vs. no aphid samples at the feeding ROI. (C) Col-0
vs. tpc1-2 control samples at feeding ROI. (D) Col-0
vs. tpc1-2 aphid exposed samples at feeding ROI.
The marker line on the x-axis (t=0) represents aphid
settling to feed. Shaded region represents a
significant different between treatments (p<0.05).
2+
A significant Ca burst was recorded between 2-24 min in Arabidopsis Col-0 reporter plants at a localised site
around the area of aphid feeding during the plant-aphid interaction. No changes in fluorescence intensity
2+
between aphid interaction and control leaves at the systemic site suggests the Ca burst is not systemic in
2+
nature. Ca bursts in TPC1 KO reporter plants appears to be reduced relative to the Col-0 control.
Mp10 knockdown M. persicae may be less capable of supressing the Ca
2+
burst in Arabidopsis
M. persicae raised on dsMp10 containing Arabidopsis show a 30-60% knockdown in the aphid effector Mp10.
2+
During the aphid-plant interaction, Mp10 knockdown M. persicae show reduced suppression of the Ca burst
relative to control, dsGFP raised M. persicae (Fig. 3).
0 min
1 min
2 min
3 min
4 min
5 min
6 min
7 min
2+
Figure 3. dsMp10 knockdown M. persicae feeding on Arabidopsis GCaMP Ca reporter plants. dsGFP raised M.
persicae are control. Shaded areas represent significant increases in fluorescence intensity (p<0.05).
2+
Ca bursts are induced in Arabidopsis in response to aphid interactions. This signal is limited to the site of
2+
stylet penetration and is not systemically propagated. The Ca response may be TPC1 dependent and further
2+
evidence that Mp10 acts to supress Ca is presented.
Future directions
Repetition of imaging data is necessary and further investigation into candidate receptors is required to
2+
2+
confirm the aphid role in inducing Ca bursts. Comparisons between Ca signals during non-compatible and
compatible interactions can be assessed using non-host aphid species.
References
[1] Bass, C., Puinean, A. M., Zimmer, C. T., Denholm, I., Field, L. M., Foster, S. P., Gutbrod, O., Nauen, R., Slater, R. & Williamson, M. S.,
(2014). The evolution of insecticide resistance in the peach potato aphid, Myzus persicae. Insect. Biochem. & Mol. Biol., 51, 4151
[2] Hogenhout, S. A. & Bos, J. I. B., (2011). Effector proteins that modulate plant-aphid interactions. Cur. Opin. in Plant Biol., 14, 42242.
[3] Pitino, M. & Hogenhout, S. A., (2013). Aphid protein effectors promote aphid colonization in a plant species-specific manner. MPMI,
26(1) 130139.
[4] Kyung-Nam Kim, K., Cheong, Y. H., Grant, J. J. & Pandley, G. K., (2003). CIPK3, a Calcium SensorAssociated Protein Kinase That
Regulates Abscisic Acid and Cold Signal Transduction in Arabidopsis. Plant Cell, 13, 411-423.
Grant impact (self)

The positive impact of the Biochemistry society summer studentship on my enthusiasm for research and
ambition to pursue a career in scientific research has been immeasurable. I feel extremely privileged to have
had the opportunity to conduct such work this summer and I am determined to build on this experience.
Value of the studentship (Dr Tony Miller)

The important data obtained by James has helped to uncover the role of several important components of the
2+
plant-aphid Ca signalling system. His work has demonstrated the role of aphid effector molecule, Mp10 in
suppressing plant defence responses. On the plant side, he has helped to demonstrate a clear role for the
2+
2+
vacuolar channel TPC1 in aphid-elicited Ca signals. The role of the plant Ca -interacting kinase, CIPK3 is
these responses is less clear. His work has provided underpinning information for a grant proposal to
investigate these components in more detail.
An investigation into the potential of antisense oligonucleotides

to reverse skeletal muscle fibrosis.
Student: James March
Supervisor: Professor George Dickson
Daily Supervision: Dr. Linda Popplewell
Background: Duchenne muscular dystrophy (DMD) is part of the muscular dystrophies group of inherited disorders,
which are characterised by progressive muscle tissue wasting and its replacement with fibrotic tissue. In DMD, this leads
to the gradual loss of strength and mobility, leading to severe disability and eventual death from cardiac and respiratory
failure. There are currently several therapeutic approaches in trial to address the genetic basis of DMD. However,
fibrosis is a major challenge for these strategies because the development of fibrotic tissue precludes a complete
restoration of muscular function. As a result, an additional strategy must be developed to address the fibrotic problem.
Fibrogenesis involves different cellular and molecular elements. One is connective tissue growth factor (CTGF), which
acts downstream of transforming growth factor type- 1 (TGF1), which is associated with the induction and progression
of virtually all fibrotic remodelling in different biological systems, including skeletal muscle. CTGF is thus a potential
target for strategies seeking to address fibrosis in DMD. It has been demonstrated that treatment with an anti-CTGF
monoclonal antibody can reduce fibrosis in the mdx mouse model of DMD (Morales et al. 2013). In addition, antisense
oligonucleotides (AOs) have been used to inhibit CTGF expression and limit fibrosis in other biological systems (Sisco et al.
2008). However, the potential of AOs to address fibrosis in muscle has not as yet been investigated.
Aim: To investigate the potential of AOs to knockdown CTGF expression in myoblasts by disrupting the CTGF transcript
reading frame via the induction of exon skipping. To evaluate this in vitro in cultured murine C2C12 myoblasts at the
transcript and protein level through nested RT-PCR and Western blot analyses.
Description of work: Human and mouse CTGF transcript sequences were interrogated in silico to identify exonic splicing
enhancer (ESE) sequence motifs within out-of-frame exons. A series of 25mer AOs with phosphorodiamidate morpholino
oligomer (PMO) chemistry with high theoretical binding energies and potential invasive nature were designed to target
relevant ESEs. Designed AOs were tested in vitro in murine C2C12 myoblasts grown at 37C, 5% CO 2 in DMEM
supplemented with GlutaMax, 4.5g/L D glucose, pyruvate, 10% fetal calf serum and 1% penicillin/streptomycin.
The effect of TGF1 on CTGF transcription over time (0, 1, 3, 6, 9 and 24 hours) and at different concentrations (0, 5, 10,
15 or 20 ng/ml TGF) was evaluated by RT PCR analysis of harvested RNA. To assess the effectiveness of designed AOs to
knockdown CTGF expression at the transcript level, a dose response (2, 5 or 10 M) was carried out, with and without
TGF1 (10 ng/ml). RNA was harvested at 24 hours or at indicated timepoints using QIAshredder and RNeasy mini kits
from QIAGEN. 500 ng of purified RNA from each sample was amplified using the GeneScript RT-PCR System (Quantig). 2
l of first round product was further amplified using Taq polymerase mastermix (Quantig). 10 l of second round product
was run on 2% agarose gels against Hyperladder IV (Bioline) with 1x SYBRSafe DNA gel stain in TBE. Densitometric
analysis of band intensity on gel images was undertaken using GeneTools software from Syngene.
The capability of the optimal AO (at 10 M) to knockdown CTGF protein expression both with and without TGF1 (10
ng/ml) was assessed. Protein was harvested following 24 hours incubation and the protein concentration of the cell
lysate determined by DC assay. Following denaturation, 50 g of protein from each sample was run on a 3-8% Tris
Acetate NuPage gel and then transferred to nitrocellulose membrane. The membrane was blocked with 4% BSA in 1x
PBS-T and then incubated overnight with 1:10000 mouse anti-vinculin (Sigma SAB4200080) and 1:1000 rabbit anti-CTGF
(abcam ab6992) primary antibodies. After repeated washing in 1x PBS-T, the membrane was incubated with 1:10000
goat anti-mouse IRDye800CW and donkey anti-rabbit IRDye680LT (Licor) for 1 hour. Antibodies were diluted using 4%
BSA in 1x PBS-T. Following washing, the membrane was analysed with Odyssey software. Bands for vinculin and CTGF
were quantified using ImageJ software and CTGF was normalised to vinculin.
Results: Results of nested RT-PCR analysis of the effect of TGF1 on CTGF transcription over time showed that
transcription levels increased from 3 to 9 hours, but decreased 24 hours after treatment, suggesting that the optimum
incubation time for inducing CTGF expression was 6-9 hours. Nevertheless, a 24 hour incubation was used in subsequent
experiments since this is minimum required for AOs to have an apparent effect in vitro. Results showed that 5-15 ng/ml
TGF1 induced similar levels of CTGF transcription. 10 ng/ml was selected for subsequent experiments. However, the
method used could not accurately determine levels of CTGF transcription. The signal of bands analysed on gels reached a
maximum level for some treatments and differences in expression levels could not be distinguished.
Of the four AOs tested, only AO1 showed evidence of exon skipping when harvested RNA was analysed by RT-PCR and
thus potential effectiveness at knocking down CTGF transcript levels in C2C12 cells (Fig. 1). When treated with 2 M
PMO1 without TGF1, the observed skipping was 7.2%. This increased to 38.4% when treated with 5 M PMO1 and
44.1% with 10 M. However, the degree of skipping was lower when cells were also treated with TGF1: at 2 M PMO1
no
skipping
was
observed, at 5 M
there
was
6.9%
skipping and at 10 M
17.2% skipping. These
results indicate that
PMO1 is effective at
inducing exon skipping
at higher doses, which
are
particularly
required
in
the
presence of TGF1.
Western blot analysis showed that 10 ng/ml TGF1 induced CTGF expression by 67.1% in C2C12 cells (Fig. 2). The blot
appears to show knockdown in expression by 10 M AO1 without TGF1. However, once normalised to vinculin, no
knockdown of endogenous and TGF1-induced CTGF
expression by AO1 was observed. This suggests that
AO1 was not effective at knocking down CTGF
expression after this incubation time at this
concentration. However, it is possible that knockdown
may be evident upon analysis of CTGF levels in culture
media since CTGF is a secreted protein.
Future directions: Quantitative PCR could be used to
quantify more accurately the effect of TGF1 on CTGF
transcription
over
time
and
at
different
concentrations, as well as the degree of exon skipping
observed with AO1. To assess more accurately the
ability of AO1 to knockdown CTGF protein expression,
the abundance of CTGF present in growth media
should be assessed by Western blot analysis in
addition to cell lysates. A further dose response
experiment could be carried out to assess what concentration is required to induce significant skipping in the presence of
TGF1. Longer AOs could also be examined as these will have higher binding energies and hence enhanced efficacy.
Departures from original proposal: Before transfection of C2C12s with AOs, the effect of TGF1 on transcription levels
over time and the effect of different concentrations of TGF1 were assessed. Endoporter was used to deliver AOs to
cultured cells instead of nucleofection. Due to time constraints, designed AOs were not tested in human RD myoblasts.
Value of the studentship to the student: The Studentship has been of immense value to me. It has afforded me
opportunity to gain experience of working in a research laboratory and allowed me to develop my laboratory skills, grow
in confidence and learn new skills. I have been able to learn from experienced researchers and discuss with them their
current projects, broadening my horizons. The experience provided by the grant has confirmed to me that my career
aspiration to work as a research scientist in the field of genetic therapy is indeed what I want to do.
Value of the studentship to the lab: This studentship has enabled the securing of important preliminary data that will
strengthen planned applications for research funding. The need for effective targeting of skeletal muscle fibrosis is
becoming more and more vital. With the necessary development and further study, the work performed within the
studentship has the potential to provide an anti-fibrotic therapy. New avenues of research for the lab have been
investigated with this studentship and it has therefore been extremely fruitful and worthwhile.
Bibliography:
1.
2.
Morales, M.G., Gutierrez, J., Cabello-Verrugio, C., Cabrera, D., Lipson, K., Goldschmeding, R. & Brandan, E. 2013, "Reducing CTGF/CCN2 slows
down mdx muscle dystrophy and improves cell therapy", Human molecular genetics; Hum.Mol.Genet., vol. 22, no. 24, pp. 4938-4951.
Sisco, M., Kryger, Z.B., O'Shaughnessy, K.,D., Kim, P.S., Schultz, G.S., Ding, X., Roy, N.K., Dean, N.M. & Mustoe, T.A. 2008, "Antisense inhibition of
connective tissue growth factor ( CTGF/ CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo", Wound repair and
regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, vol. 16, no. 5, pp. 661.
Student: Jamie Hanna

Supervisors: Dr Maelosa McCrudden and Aaron Courtenay
Investigating dissolving microneedle arrays as a vaccine delivery platform
Background:
In todays society, the emergence of microneedle (MN) drug delivery systems has had a major
impact on the potential administration of various drugs and vaccines painlessly and in a minimally
invasive manner (Quinn et al. 2014). MN delivery offers an alternative to the utilisation of
conventional parenteral delivery devices such as the hypodermic needle and syringe, which
ultimately results in nociceptive stimulation upon administration leading to much discomfort and
potential distress in numerous patients (Quinn et al. 2014). Extensive research being conducted in
this area has led to the development of five main MN subtypes; solid, hollow, coated, dissolving and
hydrogel (super-swelling), with the latter two forms being of major focus throughout the project
described herein. Delving deeper into the functionality of firstly the dissolving MN platform, the
drug/vaccine to be delivered is incorporated into the polymeric matrix of a dissolving MN
formulation, composed of either Gantrez S-97 (Polymethylvinylether/maleic acid or PMVE/MA) or
polyvinylpyrrolidone (PVP) (McCrudden et al. 2014). The heights of the needles on MN patches can
range between 50 900 m and function by imbibing interstitial fluid from the skin upon delivery
and this results in fluidisation of the polymeric chains and hence dissociation of the needles,
resulting in release of the drug across the stratum corneum, the
outermost layer of the skin and that which is most difficult to traverse
(Quinn et al. 2014).
The second MN form previously mentioned, that was majorly utilised
throughout this project, was the hydrogel subtype. Making use of
Gantrez S-97 as the main polymeric network for the MN formulation;
the formulation also contains polyethylene glycol 10,000 (PEG 10,000)
which forms cross-links with the PMVE/MA and enables the array to
swell as a result (Donnelly et al. 2014). Therefore, on insertion of a
hydrogel MN array into the skin of an individual, this results in
swelling of the array upon absorption of fluid from the skin and
thus allows for diffusion of drug from a reservoir (a lyophilised
Figure 1. Image of personally
wafer containing Ibuprofen-Sodium, for example) through the
produced hydrogel MN, using light
hydrogel system, and across the stratum corneum, along a
microscope
concentration gradient (Quinn et al. 2014). The two MN types
described in detail were utilised extensively throughout this project.
Departures from the original proposal:
It was agreed with my supervisor, Dr. McCrudden that I would be heavily involved in the day-to-day
experimental work on two distinct projects, due to the fact that the vaccine delivery project involved
some substantial lag times (e.g. time between prime and boost vaccinations etc.). To this end, my
eight week project in the lab involved two themes, namely:
Vaccine delivery via MN
Comparing and contrasting MN arrays manufactured using two different mechanisms
Vaccine Delivery Project:

To deliver an efficacious dose of a novel vaccine, which is used to induce immunisation
status to the common bacterium, Haemophilus influenzae NCTC8467 , utilising a dissolving
MN platform, via the ears of CRL Nude Hrhr elite mice, as described previously by (Zaric et al.
2015).
To determine immunoglobulin titres subsequently quantified from the mice serum (postsecondary vaccine boost) which are then compared with titres obtained when the mice
received an IM injection of the same vaccine, to determine if delivery was successful, and to
what extent.
Comparing and contrasting the production quality for both hydrogel and dissolving MN platforms
between two different manufacturers:
To obtain release data, utilising a modified Franz cell set-up, for two small molecular
therapeutics (Ibuprofen-sodium and Caffeine) through two different model membranes,
namely neonatal porcine skin and Aquacel (both of which successfully mimic the stratum
corneum), using dissolving and hydrogel MN manufactured by two different organisations
(Queens University Belfast (QUB) and a pharmaceutical partner company).
To make use of the relevant analytical techniques such as Enzyme-linked Immunosorbant
assay (ELISA) and High Performance Liquid Chromatography (HPLC) as determinant methods
for quantification of sample concentrations, which allowed for calculation of amount of
drug/protein released and subsequently the Percentage (%) release of the relevant
substances, in order to gain knowledge on whether the MNs manufactured by the different
companies will portray different release curves, as a result of differences in manufacture
technique.
To also determine if there is any significant difference in the release of pharmaceuticals/
biomolecules through the two different MN platforms being utilised (Dissolving and
Hydrogel) through comparison of release data produced for each MN type, for a given
substance.
Methods:
Hydrogel MN arrays were formulated by preparing aqueous polymer blends, casting into silicone
moulds, and thermal polymer cross linking procedure as described by (Donnelly et al. 2014).
Dissolving MN arrays were formulated by mixing aqueous polymer blends, followed by casting into
silicone moulds, and drying at room temperature, as described by (McCrudden et al. 2014).
Ibuprofen-sodium lyophilised wafers were manufactured according to a previously documented
method briefly, excipients were incorporated into a homogenous liquid, cast into moulds and
lyophilised as per protocol (McCrudden et al. 2015).
In vitro drug permeation experiments were carried out

as previously described by (Ng et al. 2010) with some
variations, including the use of two model membranes,
namely Aquacel and neonatal porcine skin.
IgG ELISA protocol, for quantifying the proportions of
immunoglobulin G levels, was conducted in accordance
with the kit provided (Mouse Anti-Hib-PRP IgG
Figure 2. A representation of a modified
(Haemophilus influenzae type b Polysaccharide,
Franz cell setup using a lyophilised wafer
PRP), ELISA Kit Cat. 980-120-PMG, ALPHA
and a hydrogel MN system, designed by
DIAGNOSTIC INTERNATIONAL, 6203 Woodlake
(Donnelly et al. 2014).
Centre Drive. San Antonio. Texas 78244. USA.).
Results and Discussion:

Vaccine Delivery Project:
Following collection of serum samples from the test mice, analysis of IgG antibody titre was
conducted using a commercially available ELISA. Concentrations of detectable IgG antibody titres are
represented in the scatter plots figure 3, below:
400
200
N
M
PV
P
Q
U
S97
M
B
Q
U
In
du
s
tr
y
Pa
rt
ne
rS
-9
7
2u
g
IM
on
tr
ol
1u
g
IM
IgG Antibody titre U/ml
600
Figure 3. Scatter plot representation of nude mice IgG antibody titre vaccinated via IM injection and
MN application of Industry partner S-97 MN, QUB S-97 MN, and QUB PVP MN containing a
Haemophilus influenzae vaccine.
Mice exposed to no treatments were kept as absolute negative controls and did not show any
detectable IgG antibody titre. The data obtained for the 10 mice subjects, whom received a 1g dose
of IM vaccine, showed that 9 of them displayed appreciable titres for the present of IgG antibodies,
which the highest and lowest titres being 237.2 and 7.9 U/ml, respectively, with an average titre of
72.9 U/ml. 3 mice were treated with 2g IM, in which 1 of them showed a positive IgG titre at 140.4
U/ml. The 4 mice which were treated with QUB PVP MN arrays, only one of them showed a positive
antibody level of 387.3 U/ml. A further 4 mice were treated with QUB S-97 MNs containing the
vaccine, in which all of them displayed positive IgG antibody titres with the highest and lowest titres
being >500 and 6.8 U/ml. the average titre was found to be 137.6 U/ml. Upon treating 4 of the mice
with MN arrays produced by the partner company containing the vaccine, none of the mice
produced detectable IgG antibody levels.
Investigating results further, the partner pharmaceutical company formulations displayed no
detectable response, which when compared to QUB MN arrays containing the vaccine, all of the
mice attained appreciable antibody titres. Upon completion of an in vitro dissolution test of all the
MN arrays utilised during the test, it became obvious that the partner companys MN arrays swelled
when immersed in 30mls phosphate-buffer saline, whereas all other arrays dissolved fully. The MN
manufacturing protocols of each of the organisations was then reviewed, and it appeared that the
partner company utilised a heating step, of 50 C for 15 minutes, during their drying stage of
manufacture, whereas QUB MNs were dried for approximately 48 hrs at room temperature. The
implications of the heating step, using high thermal energies, could result in overcoming various
activation energies of components within the formulation, resulting in the Gantrez S-97 potentially
crosslinking with the active vaccine component, and therefore trapping, and preventing release of
the vaccine from the MN array. This would ultimately result in the mice not developing a humoral
response to the vaccine, which is one theory that has been drawn from the data collected and
ultimately requires a more in depth investigation.
Comparing and contrasting the production quality for both hydrogel and dissolving MN platforms
between two different manufacturers:
Upon analysis of the caffeine permeation from both types of dissolving MNs through an
Aquacel/tinfoil model membrane (Figure 4.), QUB dissolving MNs showed percentage release of
64.3 10.9 % caffeine over 24 hours when compared to the partner pharmaceutical companys
permeation data, which showed 50.1 16.0 % caffeine permeation after 24 hours.
Statistically, on completion of the Man Whittney U test, there is no significant difference (p>0.05)
between the permeation data obtained for either company. Similar release is displayed, indicating
that there is little difference in caffeine permeation across the stratum corneum (modelled in this
study by Aquacel and tinfoil, using a modified Franz cell setup) between the MNs from the two
organisations. Therefore this indicates that the manufacturing method utilised by QUB, could be
translated to industrial manufacture for dissolving MN containing caffeine.
100
Percentage Caffiene Permeation (%)
90
80
70
60
50
Industry Partner dissolving

MN caffeine
40
QUB dissolving MN caffeine
30
20
10
0
0
500
1000
1500
Time (minutes)
Figure 4. Percentage caffeine permeation from LTS caffeine dissolving and QUB caffeine dissolving
MN through Aquacel/aluminium foil synthetic skin model on a modified Franz cell apparatus over
24 hours. n = 3 SEM.
Assessing the data obtained from the QUB vs. the large pharmaceutical partner company
permeation study (Figure 5.), utilising hydrogel MNs formulated by each company and Ibuprofen
wafers through neonatal porcine skin, there was approximately 50.1 23.0 % ibuprofen permeation
through the porcine skin using QUB MN arrays over a time period of 24 hours, compared to the
partner pharmaceutical companys MN arrays which demonstrated that 52.5 13.3 % Ibuprofen
permeation was achieved over the same period of time.
Upon further analysis of the results obtained, Mann Whitney U analysis (p>0.05)showed no
significant difference between the percentage of Ibuprofen released from either the QUB hydrogel
MN formulation or the partner pharmaceutical companys MN formulation. This indicates that the
QUB formulation could therefore be translated to industrial manufacture, to allow for up-scaled
production of these particular MN arrays. Advantages of industrial manufacture include mass
production capabilities, cost efficiencies, and ultimately advances in further MN development
processes.
Percentage Ibuprofen permeation (%)
100
90
80
70
60
Industry Partner MN + Ibu
wafer
50
40
QUB SS MN + Ibu wafer
30
20
10
0
0
500
1000
1500
2000
Time (mins)
Figure 5. Percentage Ibuprofen permeation from LTS manufactured super swelling MN and QUB
manufactured super swelling MN through neonatal porcine skin on a modified Franz cell apparatus
over 24 hours. n = 3 SEM.
Conclusion:
The antibody titre values obtained from the vaccination of mice with various MN formulations
containing the vaccine, indicated that the QUB S-97 and QUB PVP may be suitable for MN
manufacture as all mice responding with antibody production for QUB S-97 and one mouse
responding in the QUB PVP cohort, whereas the partner companys formulation of the vaccinecontaining MN arrays (S-97), there was no detectable levels of IgG antibody. This indicates that
further formulation work is required to optimise the industrial manufacturing process.
The ibuprofen permeation study through porcine skin, utilising both the QUB and a partner
pharmaceutical companys MN arrays, indicated that hydrogel arrays were developed that allowed
50.1 23.0 % and 52.5 13.3 % caffeine permeation, respectively, over a period of 24 hours when
combined with a lyophilised wafer. The Caffeine permeation study through an Aquacel/Tinfoil
model membrane, utilising both QUB and the partner companys caffeine dissolving MN arrays,
indicated that the dissolving arrays developed by QUB and the pharmaceutical company lead to 64.3
10.9 % and 50.1 16.0 % caffeine permeation, respectively. This data obtained suggests that the
QUB MN systems could be successfully transcribed and utilised in order to allow for large scale
production of both the hydrogel and dissolving MN platforms by the partner pharmaceutical
company.
Future directions of the project:

Work currently being carried out in utilising the dissolving MN platform to deliver an efficacious dose
of a novel vaccine could potentially lead to a vast number of further in vivo tests being conducted,
such as quantifying antibody levels in Guinea pigs or Rabbits, for example, on administration of
vaccine-containing dissolving MNs. If the results look promising, and this drug delivery methodology
is proving to show the development of an immune response in in vivo studies, then Phase I clinical
trials could, in time, be carried out, to test the viability of this concept in human subjects.
Value of the Studentship:

I believe that in obtaining this studentship, it has proven to be a most invaluable experience and I
have gained more from the 8 week placement than I could have hoped for. I had the opportunity to
gain continuous lab experience, which under normal circumstances throughout the normal academic
year due to scheduled lectures alongside lab classes, would not be realistically achievable. My
confidence and independence has grown drastically over the course of the placement and I now feel
more comfortable conducting experiments and analysing data sets. Ive developed my skills on a
variety of different laboratory and analytical techniques that will no doubt aid me in practical classes
during the next two years of my degree, for example, how to carry out permeation studies utilising
modified Franz cells, how to carry out ELISA, characterisation of MN arrays, how to manufacture
both dissolving and hydrogel type MN and also how to analyse HPLC data obtained from collected
samples. I have also undoubtedly gained new knowledge on topics at the forefront of groundbreaking research which I found will benefit me during the remaining course of my degree.
On completion of the 8 week summer placement, I have definitely gained a greater insight into the
field of scientific research and into what it could potentially be like to be studying for a PhD. I have
immensely enjoyed my experience here in the lab and I definitely will likely endeavour to undertake
a PhD post-graduation, potentially in an industry-related setting, as it will, of no doubt, be a very
rewarding experience and will benefit in my future career progression.
From the supervisor: Dr. Maelosa Mc Crudden
I firmly believe that undergraduate vacation studentships are a very valuable experience for both the
student who undertakes the research project but also the research team in which the work is
undertaken. With this in mind and as explained by Jamie previously, this studentship has offered him
a wonderful opportunity to carry out ground breaking research that simply cannot be covered in
routine undergraduate practical classes. Jamie has been an excellent addition to the team in our
laboratory and has been a very conscientious and enthusiastic student. He was very motivated and
after undergoing training in various methodologies, he became proficient and independent in his
approach to his work. I am confident that Jamie will excel as he continues his MPharm studies and I
would like to personally thank the Biochemical Society for funding Jamies work. I believe that it was
a worthy investment in this talented young student.
References:
Donnelly, R.F. et al., 2014. Hydrogel-forming microneedles prepared from super swelling polymers
combined with lyophilised wafers for transdermal drug delivery. PloS one, 9(10), p.e111547.
Available at:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4216095&tool=pmcentrez&rende
rtype=abstract [Accessed August 16, 2015].
McCrudden, M.T.C. et al., 2015. Considerations in the sterile manufacture of polymeric microneedle
arrays. Drug delivery and translational research, 5(1), pp.314. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/25787335 [Accessed August 18, 2015].
McCrudden, M.T.C. et al., 2014. Design and physicochemical characterisation of novel dissolving
polymeric microneedle arrays for transdermal delivery of high dose, low molecular weight
drugs. Journal of controlled release: official journal of the Controlled Release Society, 180,
pp.7180. Available at:
rtype=abstract [Accessed July 5, 2015].
Ng, S.-F. et al., 2010. Validation of a static Franz diffusion cell system for in vitro permeation studies.
AAPS PharmSciTech, 11(3), pp.143241. Available at:
rtype=abstract [Accessed August 14, 2015].
Quinn, H.L. et al., 2014. The role of microneedles for drug and vaccine delivery. Expert opinion on
drug delivery, 11(11), pp.176980. Available at:
Zaric, M. et al., 2015. Dissolving microneedle delivery of nanoparticle-encapsulated antigen elicits
efficient cross-priming and Th1 immune responses by murine Langerhans cells. The Journal of
investigative dermatology, 135(2), pp.42534. Available at:
Student: Jennifer Shoesmith

Supervisor: Professor Alison Baker and Dr David Carrier
Gaining structural insights into the plant ABCD transporter COMATOSE through Cysteine
Probing
Introduction
Comatose (CTS) is a plant peroxisomal membrane
protein (From Arabidopsis thaliana) and is an ABC
transporter (1). CTS functions in many plant
developmental and metabolic processes. It has
been shown that CTS transports long chain fatty
acids and some hormone precursors into the
peroxisome to undergo -oxidation (1, 2). CTS
appears to transport Acyl-CoAs and has a thioesterase activity (3). CTS is a large hydrophobic
membrane protein (4), and therefore traditional
structural techniques like X-ray crystallography are
challenging for determining its structure. CTS
contains 10 cysteines in total (5), and their
accessibility can be probed with Cys reactive
reagents e.g. maelimide biotin (6) allowing
information to be gained about the accessibility of
particular cysteines (7). By selectively mutating
individual cysteines and combining results with
information from a homology model structutal
information can be obtained. To ensure mutants are
functional they are tested for the ability to
complement S.cerevisiae Pxa1/Pxa2 for growth
on oleate (8).
Aims
To test the importance of individual cys in
COMATOSE with a view to creating a cysless
mutant and reintroducing at various sites for
structural studies.
To assess the functionality of cys mutants in yeast.
To use cysteine mutants in maelimide binding
experiments to elucidate structural information
using mass spectrometry.
Methods
Cysteine mutants were generated using single
primer reactions in parallel mutagenesis (9) and
traditional site directed mutagenesis(10). Plasmids
were maintained in Omnimax cells grown on LB
ampicillin and prepared using Promega wizard kits.
Plasmids were transformed into yeast cells by
treatment with lithium acetate and heat shock for
30mins at 42c and spread on SD-agar with
appropriate drop out amino acids for selection (11).
Oleate growth assays performed according to
methods in (12).
To correct deletions in mutated plasmids overlap

extension PCR was used (13).
All sequencing was performed by Beckmann
Coulter Genomics.
HA and His tagged halves of the CTS protein were
also transformed into Pxa1/Pxa2 yeast cells,
from these cells a light mitochondrial fraction was
produced containing peroxisomes (14). Coomassie
protein gels and a western blot were used to
determine the presence of CTS in the fraction.
Results and conclusions

Mutants C608S, C1118S and C1180A were
successfully
generated
using
mutagenesis,
confirmed by sequencing. These mutants were then
transformed into yeast cells containing the other
(wild type) half of the protein for functional studies.
Other mutants that had already been generated
were tested for function by growth on oleate.
According to the growth assay fig 1, C55S, C156S,
C245S, C1118F, and C1278S all grew on the oleate
plates therefore function has not been compromised
by the mutation. C608G did not grow in the oleate
assay therefore function has most likely been
compromised by this mutation. Tagged halves of
the CTS protein were transformed into yeast. A light
mitochondrial fraction was taken from these cells
and run on SDS and probed by western blot. There
was a signal for Anti-HA antibody as well as AntiHis antibody suggesting that both halves are
present in the fraction and not in cytosolic
supernatant Fig 2.
Discussion and future directions

The cys mutants that had not yet been produced
were C608S, C768S, C1118S and C1180A.
Mutagenesis was performed with appropriate
primers and plasmids were propagated and
sequenced. The first set of colonies which was
sequenced all were WT.
The mutagenesis
experiments were repeated using the single primer
method and traditional mutagenesis. The traditional
mutagenesis experiment gave a better level of
plasmid amplification so these reactions were used
to transform cells. These plasmids were also sent
for
sequencing.
According
to
Fig 1, oleate growth assay. Yeast mutants were grown on SD-low glucose to acclimatise to using a different carbon
source. Cells were grown overnight in YPO. Cells were collected by centrifugation and re-suspended in sterile water.
Cells were diluted to the same OD600 and streaked on oleate plates. A, negative control, growth is due to
contamination no yeast present. B, positive control many yeast cells is some contamination. C, C55S some growth
observed. D, C156S some growth. E, C245S large amount of growth. F, C608G no growth. G, C1118F some growth. H,
C1278S large amount of growth.
Fig 2. SDS Page and western blot of light mitochondrial

fraction. A, coomassie stained gel. 1 molecular weight ladder, 2
supernatant 5L, 3 pellet 5L, 4 supernatant 10L, 5 Pellet
10L, 6 supernatant 15L, 7 pellet 15 L, 8 pellet 20 L,9
supernatant 20 LB western blot of light mitochondrial fraction
to detect pseudo-halves of CTS. 1 and 4, molecular weight
ladder. 2 supernatant with Anti-HA no protein present. 3 pellet
with Anti-HA Small band between 46kDa and 35kDa N-terminal
half is present in the light mitochondrial fraction as expected
although not at the expected size. Full length CTS is 149,576,
and the half size ~70kDa. 5 supernatant with Anti-His no
protein present. 6, pellet with Anti-His 2 bands present
therefore C-terminus is also present in the light mitochondrial
fraction, although bands are also not at the expected length.
agarose gels the Dpn1 digest to remove parent

plasmid was successful which was backed up by a
transformation with Dpn1 digested plasmid where
no colonies were observed. Further colonies from
original mutagenesis were grown and the plasmids
sent for sequencing.
C768S still needs to be generated as all sequencing
came back as wild type.
C631S previously generated by another student
also contained a secondary mutation A412V,
primers were designed to remove this mutation and
mutagenesis was performed. The plasmid was

propagated in E.coli and the plasmid sequenced.
However this mutagenesis came back still
containing A412V and will require repeating.
C1115-1p had a 45bp deletion upstream of the
desired mutation, to correct this overlap extension
PCR was performed. The first step PCR
amplification of the correct sequence was
successful, however the second step correction had
no amplification of the plasmid.
Mutants of C608S, C1118S and C1180A were
successfully generated, these plasmids were then
used to transform yeast cells containing the other

respect half of the protein for functional assay.
Mutants yeast cells were grown on oleate as a
functional test. Most of the existing mutants
displayed some growth on the oleate media as seen
in fig 3. C55S, C156S, C245S, C1118F, and
C1278S were all able to grow on the oleate media
meaning these mutants have retained function to
transport fatty acyl-CoA across the peroxisomal
membrane. C608G had no growth on the oleate
plates suggesting that the function of this mutant
has been disrupted and is unable to transport fatty
acyl CoAs across the peroxisomal membrane.
However this experiment is only a qualitative
measure of function. A better test of function would
be to directly measure the levels of -oxidation in
mutant cell peroxisomes. The other mutants
generated also require testing for function C608S,
C1118S and C1180A.
For structural studies tagged halves of CTS were
transformed into Pxa1/Pxa2 yeast. These cells
were grown in low glucose to acclimatise to the
oleate media without glucose. This culture was
used to inoculate 1L of YPO in which the cells were
grown overnight. This culture was used to generate
a
light
mitochondrial
fraction
containing
peroxisomes. This fraction was run on a SDS PAGE
and blotted with anti-His and anti-HA antibodies to
detect each half of the protein. Figure 2 shows that
both halves of the protein are present in the light
mitochondrial fraction and not the cytosolic fraction.
The protein is correctly localised. However bands
are not observed at the expected length for CTS full
or half size. This may be due to degradation during
the purification process which CTS is susceptible to.
To test if the bands contain degraded CTS they can
be cut out and used in mass spec. These cells
expressing tagged halves of CTS can now be used
for purification of CTS for structural studies using
mass spec.
many existing skills like mutagenes, E.coli culture

and manipulation, DNA gels, protein gels and
western blots. I have learnt how to troubleshoot
various experiments when they are not working.
The studentship has furthered my aspirations to
continue my study to PhD level and undertake
further research.
Value of the Studentship for the lab.
The studentship has allowed us to obtain 3 new
mutants and re-test some existing ones. This is a
good outcome for only 8 weeks. It has also allowed
us to start developing the methodology for probing
the accessibility of the cys residues, something we
intend to take forward in future. Jenny has been a
great asset to the lab and the techniques she has
learned will help her in her M.Biol project with us in
the upcoming academic year.
References
1.
Theodoulou, F.L. et al. Peroxisomal ABC

transporters. FEBS Lett. 2006, 580(4), pp.113955.
2.
Theodoulou, F.L. et al. Jasmonic acid levels are

reduced in COMATOSE ATP-binding cassette
transporter mutants. Implications for transport of
jasmonate precursors into peroxisomes. Plant
Physiol. 2005, 137(3), pp.835-40.
3.
De Marcos Lousa, C. et al. Intrinsic acyl-CoA

thioesterase activity of a peroxisomal ATP
binding cassette transporter is required for
transport and metabolism of fatty acids. Proc
Natl Acad Sci U S A. 2013, 110(4), pp.1279-84.
4.
Rea, P.A. Plant ATP-binding cassette

transporters. Annu Rev Plant Biol. 2007, 58,
pp.347-75.
5.
Hayashi, M. et al. Ped3p is a peroxisomal ATPbinding cassette transporter that might supply
substrates for fatty acid beta-oxidation. Plant
Cell Physiol. 2002, 43(1), pp.1-11.
6.
Hansen, R.E. and Winther, J.R. An introduction

to methods for analyzing thiols and disulfides:
Reactions, reagents, and practical
considerations. Anal Biochem. 2009, 394(2),
pp.147-58.
7.
Kim, Y. et al. Efficient site-specific labeling of

proteins via cysteines. Bioconjug Chem. 2008,
19(3), pp.786-91.
8.
Nyathi, Y. et al. The Arabidopsis peroxisomal

ABC transporter, comatose, complements the
Saccharomyces cerevisiae pxa1 pxa2Delta
mutant for metabolism of long-chain fatty acids
and exhibits fatty acyl-CoA-stimulated ATPase
activity. J Biol Chem. 2010, 285(39), pp.29892902.
9.
Edelheit, O. et al. Simple and efficient sitedirected mutagenesis using two single-primer
reactions in parallel to generate mutants for
protein structure-function studies. BMC
Biotechnol. 2009, 9, p.61.
Departures from original proposal.

Due to time constraints and issues with yeast
transformations the maelimide biotin experiments
were not performed. However the basis for these
experiments in having the tagged halves of CTS in
yeast cells is a good starting point for purification
and mass spec. The mutants that were generated
were also not tested for function due to time
constraints.
Value of the studentship to the student

The studentship has allowed me to learn many
techniques that I have not had the chance to learn
in my undergraduate degree such as yeast culture
and manipulation and how to purify organelles from
yeast. The studentship has allowed me to practise
10.
Liu, H. and Naismith, J.H. An efficient one-step

site-directed deletion, insertion, single and
multiple-site plasmid mutagenesis protocol.
BMC Biotechnol. 2008, 8, p.91.
11.
McDonald, P.N. Two-hybrid systems. Methods

and protocols. Introduction. Methods Mol Biol.
2001, 177, pp.v-viii.
12.
Marchi, E. and Cavalieri, D. Yeast as a model to

investigate the mitochondrial role in adaptation
to dietary fat and calorie surplus. Genes Nutr.

2008, 3(3-4), pp.159-66.
13.
Bryksin, A.V. and Matsumura, I. Overlap

extension PCR cloning: a simple and reliable
way to create recombinant plasmids.
Biotechniques. 2010, 48(6), pp.463-5.
14.
Distel, B. and Kragt, A. Purification of yeast

peroxisomes. Methods Mol Biol. 2006, 313,
pp.21-6.
Biochemical Society Studentship Report

Jennifer Wood
PROJECT AIM:
The project aim was to culture A549 cells, extract proteins using a dounce homogenizer, add CO 2
(C12 and C13 labelled) to the sample, transfer the sample into a pH stat (pH 7.4), add
(trimethylsilyl)diazomethane to enable CO2 binding to protein amine groups for carbamate formation,
dialyse the sample, and digest the sample proteins into peptides using heat, dithiothreitol,
iodoethane, and trypsin. The sample peptides were then pre-fractionated using stagetips,
fractionated using a mass spectrometer to identify peptides bound to CO2, analysed on databases to
reveal the original proteins, and investigated for the role these CO2-binding proteins had in
physiological and pathophysiological processes.
PROJECT REPORT (DEVIATIONS INCLDUED):

Introduction: CO2 is a vital molecule in mammalian, bacterial, and plant cells but its protein-binding
mechanisms and roles are poorly understood due to limited appropriate laboratory
equipment/techniques; our team developed a protocol able to investigate CO 2 molecular interactions.
This project used the protocol for CO2-binding protein identification in A549 adenocarcinoma human
alveolar basal epithelial cells, hoping to reveal CO2-binding proteins and their role in physiological
and pathophysiological states for therapeutic purposes.
Method: The protocol was used to generate three C12 and C13 mammalian A549 CO2-trapped
peptide samples. The steps included cell growth (1), splitting (2), harvesting (3), lysis (4), CO 2trapping (5), dialysis (6), digestion (7), and mass spectrometry (8).
1. Transfer 10mL DEMM media into 15mL falcon tube with A549 live stock cells, centrifuge
(1200rpm/3minutes), discard supernatant, add 10mL media and resuspend pellet, transfer to
T25 flask, and incubate (37C/~3days).
2. Remove media, wash in sterile PBS x 2, add trypsin and incubate (37C/3minutes), remove
trypsin, wait until mobile (view under microscope), add 10mL of DEMM and resuspend cells,
add 4mL into T75 flask x 2, add fresh DEMM into T25, T75, and T75 flasks, incubate (37C/~3
days), remove media, wash in PBS x 2, add 10mL of PBS and scrap cells into buffer, and
centrifuge cells (1200rpm/3minutes).
3. Centrifuge cells in 25mL PBS x 3 (1000g 4C/10minutes), resuspend in 1mL hypotonic lysis
buffer, incubate for 10 minutes on ice, dounce homogenise cells (40 strokes) and view lysis
under microscope, centrifuge sample (4C 1500g/15minutes), resuspend pellet in 2mL
reaction buffer, and read peptide concentration using nanodrop.
4. Weigh out 6.8mg of C12 or C13 CO2 and add to reaction, weigh out 3x80mg TEO into
separate vials, add reaction to pH stat, and add each TEO reagent with 300uL water.
5. Microwave dialysis tubing for 5 minutes, add clips and reaction to tubing, add to 1L beaker
water, and stir overnight.
6. Collect dialysed samples in 15mL falcon tubes, centrifuge (5500g for 5 minutes), remove
supernatant, resuspend in 1mL 100mM ammonium bicarbonate buffer, transfer 100uL into
Eppendorf for digest, add 1uL 10 % SDS, add 2uL DTT, incubate at 80 C for 15 minute, add
5uL iodoethane, incubate at room temperature in dark for 30minutes, heat trypsin aliquots
from freezer at 37 C, add trypsin to each digest, incubate at 37 C/20 hours, and take to
technician and perform mass spectrometry.
Results & Discussion: Unfortunately, the A549 cells did not yield peptide results following mass
spectrometry. It was hypothesised that cellular lysis may be responsible and thus a new douncing
method using a hypotonic cellular buffer, incubation on ice, and centrifugation was used. The sample
protein concentration was also estimated using a nanodrop. The nanodrop revealed that the problem
was more likely due to the TEO CO2-trapping agent causing massive protein aggregation. During the
digestion process, addition of ammonium bicarbonate helps trypsin function, SDS aids dissolving
protein (thus removing precipitate), DTT binds cysteine residues for disulphide bridge breakage,
iodoethane maintains this bond breakage, 80C and 32C incubations denature protein, and trypsin
digests the protein; consequently, protein aggregation should not have been an issue. Therefore, it
seems the problem lay prior to digestion. For instance, when removing the sample from the pH stat
into test tubes, the aggregates frequently became lodged on the side of the pH stat, thin glass
pipette, or plastic pipette tips and were consequently lost. Thus, only a small amount of aggregated
protein (if any) remained prior to dialysis. Furthermore, digestion was performed using only a
relatively small amount on the dialysis sample. Therefore, the very low amount of protein in the
sample prior to dialysis, small amount of sample used for digestion, and potential peptide loss in prefractionation methods prior to mass spectrometry must have caused complete peptide absence from
the mammalian samples before mass spectrometry. This protocol was also performed on E.coli to
identify the issue, which did yield peptides following digestion. It seems likely that there were many
more bacterial cells and thus proteins at the start of the experiment, which is thus likely to maintain
protein within the samples even following TEO-induced aggregation.
FUTURE PROJECT DIRECTION AND PROJECT ACHIEVEMENTS:

It is hypothesised that the peptide absence during the project was due to the CO2-trapping agent
(TEO) causing massive protein aggregation and thus precipitant formation that is subsequently lost
due to handling difficulties in combination with the small volume of the sample used. E.coli and plant
cells yielded peptides using the same protocol. Therefore, it seems likely that the protocol is effective
for CO2-binding protein identification, however, alternations must be made when performing it on the
A549 cells. A higher quantity of mammalian cells used per experiment and methods that improve
sample transfer during protocol steps will both help to prevent protein loss. This will enable
mammalian CO2-binding protein identification by mass spectrometry that may potentially have
abundant therapeutic applications.
I have undertaken numerous practical sessions for my Biomedical Science course, however, these
short, pre-planned, outlined practical sessions did not seem to reflect most applied laboratory
research. Currently, my main interest involves carcinogenesis and cancer metastasis, understanding
mechanisms and signalling pathways that enable mutated cells to form a primary tumour and migrate
to distinct locations. Working on A549 CO2-binding proteins in attempt to investigate their cellular
pathways has given me insight as to how on going novel research is approached, ultimately inspiring
me to continue the pursuit of scientific-related research. Furthermore, I have gained many skills
throughout this internship. Applying for the Biochemical Society Studentship helped me learn about
funding application processes that will aid for future masters or PhD grants. Furthermore, I obtained
many transferable research skills such as flexibility and adaptability. Unforeseen issues arose during
the project and thus adjusting, learning new techniques, and attempting new and improved methods
was vital for project continuation. This also strengthened my time management skills. The project
involved generating numerous samples, which had numerous steps requiring 24 hours to complete.
Therefore, deviating from the project plan required time considerations that involved generating
multiple samples simultaneously without rushing the project. Another important achievement made
was gaining confidence for interpersonal communication. This project required working in several
laboratories with various individuals to use specific scientific equipment. I learnt how to confidently
create a professional relationship with peers and professors, asking questions when necessary. The
project improved my general communication skills; effectively explaining the project protocol and
procedures, issues, and attempts to overcome these issues. In addition, the most important
achievement was having discussions and proactive debates with others to hypothesise the peptide
absence causation, which led to E.coli experiments that helped narrow down the potential error
origin. Finally, I became familiar with numerous important scientific techniques that will have use in
many different research environments.
Membranes, Amyloid-Beta and Human Cystatin C

Background:
Alzheimers disease, a form of dementia, is a complex neurodegenerative disorder characterised by the
extracellular deposition of insoluble plaques of amyloid-beta, the presence of intracellular fibrillary tangles
and neuronal atrophy. The Alzheimers Society estimate that around 850,000 UK residents are currently
suffering from dementiai, with the cost of care at a substantial 26 billion a year. As a disease predominantly
affecting the elderly, a continued increase in life expectancy makes our mission to understand its
pathogenesis, and potentially develop treatments to avert it, all the more important.
Toxic, soluble assemblies of Amyloid-beta1-42 are believed to be key in the pathogenicity of Alzheimers
disease. Importantly, a cerebrospinal-fluid protein, cystatin C, exhibits a protective effect ii; preventing fibre
formation and regulating the toxic effect of oligomersiii. The nature of this interaction is of great interest and
is arguably most important at the membrane surface where binding is much tighter than in bulk solution.
Additionally, both peptide-membrane interactions, and the local environment at a membrane surface have
been implicated in facilitating assembly of A oligomers. A review by Relini et al. iv explores current
research into these facets of membrane surfaces, considering the two-dimensional lipid-water interface and
key membrane components such as GM1 and cholesterol to be instrumental in creating nucleation sites
and thus promoting the formation of higher order structures. Considering this, an investigation of the
interaction between A and hCC on a model membrane will likely provide a detailed insight into the
modulating effect of hCC.
Aims:
To use atomic force microscopy to characterise the behaviour of the A peptide at a membrane surface and
to investigate the ability of hCC to modulate this behaviour.
Work Carried Out:
Human Cystatin C Preparation:
BL21 cells were transformed with an IPTG inducible hCC plasmid, with the hCC protein targeted to the
periplasm. These cells were later used to inoculate 5l of M9 minimal media-Amp for growth in baffled
flasks. Cells were grown to an OD of ~0.6 and hCC expression was induced by addition of IPTG. Cells were
lysed by resuspension in 28ml EDTA following centrifugation and resuspension in 20% sucrose solution.
After another round of centrifugation the supernatant was collected and dialysed in 5l cold sodium
phosphate buffer pH7.4
Anion exchange chromatography, and size exclusion chromatography were used to obtain a suitably pure
hCC sample, as determined using SDS-PAGE analysis and size-exclusion hplc (see figure 1).
A n a ly s is o f P u r if ie d h C C u s in g H P L C
Figure 1:
1 .0
F ib B u ffe r
hCC
Abs
0 .9
0 .8
0 .7
0 .6
0 .5
0
20
40
T im e ( m in s )
60
80
High performance liquid

chromatography readout for
our purified human cystatin C
protein. A fibrillisation buffer
was run down the column to
provide the baseline shown in
pink. The orange line indicates
our hCC sample. The cystatin C,
as expected, was eluted after
38 minutes and the single sharp
peak confirms the absence of
contaminants and therefore a
good purification of the
protein.
Vesicle Preparation for Dye-leakage Assay:

Stock lipids, solubilised in chloroform, were mixed to relevant concentrations in a round-bottomed flask and
the chloroform was evaporated off under a gentle nitrogen stream. The resulting lipid film was flash frozen
in liquid nitrogen and lyophilized to remove any remaining solvent. The film was then resuspended in 1ml
5-(6)-Carboxyfluorescein by vigorous vortexing for at least 15 minutes to create Carboxyfluorescein loaded
multilamellar vesicles. These were extruded through an Avanti mini-extruder containing a 200nm membrane
to create large unilamellar vesicles and run down a PD-10 size exclusion column to remove excess
carboxyfluorescein.
A Oligomer Preparation:
A dry 0.1mg stock of A was resuspended by sonicating in 140l of 10mM NaOH for 30mins. This is then
diluted twice in 10mM HEPES buffer and the pH was adjusted to 7.4 by the addition of HCl. The resulting
suspension was then refrigerated for 24 hours. Before use, the preparations were centrifuged to remove
preformed fibres.
Transmission Electron Microscopy:
Carbon coated grids were glow discharged to create a hydrophilic surface. These were then coated in 10l
samples of oligomer preparations and negatively stained using urinyl-formate. The grids were imaged using
TEM to confirm successful oligomer formation.
Atomic Force Microscopy:
GM1, Cholesterol and DMPC were spin-coated onto a silicon wafer at 1000-1500rpm. Elipsometry was
used to characterise the thickness of the coating and showed a consistent value of ~5nm, indicating the
presence of a bilayer.
Oligomer preparations were buffer exchanged into water and 50l added to the bilayer. The sample was
allowed to sit for ~5mins before the surface was blow-dried with nitrogen. The same preparation was used to
produce samples in the absence of a bilayer on mica. The samples imaged included bilayers, oligomers on
mica and oligomers on a bilayer.
Samples were imaged using tapping mode AFM in air on a Bruker Dimension 3100 NSIV and an Asylum
MFP 3D microscope. [The tips used had a resonant frequency 320kHz and a spring constant of 42Nm-1.]
Departures from the Original Proposal:
The unforeseen breakdown of the Atomic force microscope left us behind schedule as both me and my
supervisor had to be retrained on a different machine. However, this was beneficial for me as I now have
experience with two different AFM machines and consequently two types of imaging software.
Due to numerous challenges encountered during AFM sample preparation we were unable to confidently
identify A oligomers occupying the surface of a lipid bilayer. However, as shown in the results, we were
able to image these on mica.
The dye-leakage assay proved an interesting addition to the project, especially as we encountered numerous
complications and the results obtained were unexpected. As a result I was faced with real-world laboratory
problems which challenged me in a way that undergraduate teaching labs could not.
Results and Discussion:

The preparation of A oligomers for use in both the dye-leakage assay and AFM was consistently
successful, with a variety of oligomeric species being produced (see figure 2).
Figure 2 A (left) and B (right):

Transmission electron microscopy images of the A oligomer preparation. Where A= 11500X magnification and B= 21000X
magnification. The grid was densely populated with various oligomeric species; predominantly globular oligomers and
extended worm-like oligomers. The extended oligomers are consistent with the appearance of amyloid-beta derived
diffusible ligands seen in the literature.
The initial preparation of the model membrane on silicon wafers was also reliable and a characteristic result
is shown in figure 3. The width of the constructed bilayer was consistently within 5-6nm.
Figure 3:
Model bilayer composed of DMPC,
Cholesterol and GM1. A 5 micron height
retrace of the model bilayer, imaged using
atomic force microscopy. The purple region
indicates good bilayer coverage whilst the
darker regions indicate patches of bare
silicon. Small areas coloured orange are
due to the stacking of two bilayers.
Unfortunately it was not possible to fully maintain the

integrity of the bilayer once the oligomer preparation
was added. Numerous approaches to the sample
preparation were tested but none were particularly
successful. As a result, we were unable to
conclusively identify the presence of oligomers on
our model membrane. The oligomers were instead
imaged on mica (see Figure 4) giving a result
comparable to our EM image, with the same globular
oligomers easily identifiable. The extended wormlike oligomers seen in the EM image are also present
although they are less easily distinguishable.
Figure 4: A oligomers on mica. A phase retrace of
oligomers on mica showing similar species to those
observed using transmission electron microscopy
The dye-leakage assay was unsuccessful due to problems with osmotic balance and an as yet unexplained
quenching of the fluorescence signal observed upon the addition of A oligomers to the carboxyfluoresceinloaded vesicles. The protocol was subject to numerous amendments throughout the placement but we were
unable to completely eliminate dye-leakage in the negative control (vesicles on their own) or prevent the
quenching effect that was being observed.
Future Directions:
Once the dye-leakage assay is optimised, it can be used as a reliable test for the toxicity of oligomer
preparations prior to AFM imaging.
Imaging in water is a natural progressive step for the AFM work and will likely be key in further
investigation of the effects of hCC on A assemblies at the membrane. There is also an alternative to spincoating that will be trialled, involving the adsorption, rupturing and rolling out of vesicles to give a bilayer
for use as a model membrane. In this case it will be possible to add oligomeric A to the vesicles prior to
bilayer formation, thereby eliminating the problematic step of adding the oligomers as a wet sample to an
already intact, although both will be attempted.
Both circular dichroism and NMR spectroscopy are expected to feature heavily in further investigation of
the interaction between A and hCC.
Outcomes of the Studentship:
This project has been an invaluable experience and has introduced me to a huge range of techniques and
equipment that I would otherwise have not encountered during my degree course. The first-hand research
experience has given me confidence in my decision to pursue a PhD, whilst attending weekly lab meetings
introduced me to fields of research that have inspired specific interests and will help guide my choice of
PhD project. I would like to thank the Biochemical Society for this opportunity and everyone in the NMR
group for being so welcoming and supportive throughout my placement.
Summary of Results:
We produced recombinant human cystatin c protein from a genetically engineered bacterial culture with the
intention of examining the influence of this protein on the behaviour of amyloid-beta at a model cell
membrane. The model membrane was composed of lipids in a ratio representative of the natural cell
membrane and a solution of these lipids was spin-coated onto a silicon wafer; resulting in the formation of a
membrane on the silicon surface. Amyloid-beta was incubated in conditions that allowed small clusters
called oligomers to form and it is these that were added to the membranes. The oligomers were left to
interact with the cell membrane before the sample was dried for imaging with atomic force microscopy in
air. Unfortunately we were unable to optimise our sample preparation and as a result we didnt quite get
around to adding the human cystatin c. However, I am confident that the research work carried out in my 8
weeks will allow this very shortly.
i
Alzheimers Society (2014). Financial Cost of Dementia.

http://www.alzheimers.org.uk/site/scripts/documents_info.php?documentID=418
nd
Accessed 22 August 2015
ii
Kaur, G. and Levy, E. (2012). Cystatin C in Alzheimers disease. Frontiers in Molecular Neuroscience. 5: 79
iii
Tizon, B. et al. (2010). Cystatin C Protects Neuronal Cells from Amyloid--induced Toxicity. Journal of Alzheimers Disease. 19,
885-894
iv
Relini, A., Marano, N., Gliozzi, A. (2013). Probing the interplay between amyloidogenic proteins and membranes using lipid
monolayers and bilayers. Advances in Colloid and Interface Science. 207, 81-92
Student: Laura Bruce

Supervisor: Neil Kad
Creating a nucleosome array for single molecule investigation

Background:
The development of single molecule techniques has allowed
for in-depth investigation into proteins and DNA and how
they interact with each other and other molecules that they
may encounter within a cell. Of particular interest within the
Kad lab is how DNA repair proteins locate damage in the
genome. Using a single molecule technique known as DNA
tightroping it is possible to directly image these interactions,
these investigations have shown that proteins find their
targets sites on DNA through a range to different
mechanisms[1], however it has not yet been discovered what
need there is for these multiple pathways.
The eukaryotic genome is structured in a very organised way
and there are many mechanisms in place to create a compact
structure so that large amounts of DNA can be stored
within the cell. The organisation of the DNA involves
histone proteins that form a complex with DNA to create
nucleosomes. These nucleosomes are arranged as beads on
a string, as seen in figure 2, would act as a roadblock to
the movement of proteins along the DNA
Aim:
To test the hypothesis that proteins use jumping as a
means to negotiate physical boundaries created by the
structural organisation of DNA and to understand the
impact of roadblocks on the ability of proteins to find their
target sites. To achieve this, arrays of nucleosomes on
DNA tightropes must be created, and single molecule
techniques used to image how proteins involved in DNA
repair are impeded by these structures.
Figure 1. 147 bp of DNA is

wrapped 1.65 times around
the core histone complex to
form a nucleosome[2]
Figure 2. (A) Chromatin
isolated directly
from an interphase nucleus appears in the
electron microscope as a thread 30 nm
thick. (B) This electron micrograph
shows a length of chromatin that has
been experimentally unpacked, or
decondensed, after isolation to show the
nucleosomes. (A, courtesy of Barbara
Hamkalo; B, courtesy of Victoria Foe.)
[3]
Work carried out:

Xenopus laevis histones H2a, H2b, H3 and H4 held in
PET3a vectors were received, these were the original
histones used by K. Luger et al[2]. These histones were
transformed into BL21, TOP10 and XL10 GOLD
competent cells and then the plasmid DNA isolated. These
transformations were carried out in 3 separate competent
cells as there was an issue with achieving the desired
concentrations of plasmid DNA to perform sequencing.
After isolating the DNA, it was digested with the restriction
enzymes NdeI and BamHI. This was necessary to facilitate
the subcloning of the histones into a vector that would
incorporate a poly-histidine tag (histag). PET28b was
Figure 3. Plasmid map of PET3a

showing the NdeI and BamHI
restriction sites. [4]
selected as the recipient vector because an in-frame histag sequence was accessible using the
restriction enzymes BamHI and NdeI that were also present in PET3a.
PET3a vectors containing H2a and H2b were selected and digested, the products were run on
Figure 4. Plasmid map of PET28b showing the NdeI and BamHI restriction sites and the in frame histag. [5]
an agarose gel to extract the inserts containing the H2a and H2b genes. PET28b was also
digested and run on an agarose gel, the vector was gel extracted. If H2a and H2b inserts had
been obtained the H2a and H2b would have been ligated into the PET28b and then
transformed into competent cells.
Results:
Histone
DNA concentration (ng/ul)
H2a
124.5
H2b
1.5
H3
1.9
H4
2.5
Figure 5. Concentration of DNA after transformation into BL21 cells.
Histone
H2a
22.5
H2b
23.4
H3
23
H4
34
Figure 6. Concentration of DNA after transformation into TOP10 cells.
Histone
H2a
53.7
H2b
17
H3
22.6
H4
14.6
Figure 7. Concentration of DNA after transformation into XL10 GOLD cells.
Figure 8. Agarose gel. Lane 1: digested PET28a. Lane 2: digested PET3a containing H2a.
Lane 3: digested PET3a containing H2b. Lanes 4, 5 and 6 are repeats of lanes 1, 2, and 3. The
weights of DNA indicate the amount of DNA run on each lane.
Figure 8 showed a large enough band of digested PET28a for gel extraction. No insert was
seen here but the insert was not required and was expected to only be a few bases long. No
band was seen in lane 2, even though an extremely high concentration of DNA was run on
the gel, this indicated that the high concentration seen for H2a in BL21 vectors was most
likely caused by a contaminant. Lane 3 showed a band, but no insert. The insert was the part
of the vector containing the H2b so this could not be used.
Techniques and skills acquired during studentship:

-Making agar plates and growth media
-Microbiology
-Making and accurately measuring solutions
-Isolating plasmid DNA
-Gel extraction of DNA
-Making agarose and acrylamide gels
-Appreciation of single molecule approaches
-Planning and executing experiments
-Time management
Value of Studentship:
This studentship has given me confidence in the lab, it has also taught me skills that I hope to
use if I undertake a Masters or PhD program. I have also learned a lot about reading and
researching on an individual topic and have gained a detailed understanding of the molecular
biology required in creating a nucleosome array. I have also undertaken a lot of reading on
single molecule techniques and have a greater understanding of the research being undertaken
at the moment in this field. I was interacting with the PhD students in the Kad lab on a daily
basis and experienced some of the research that is taking place in the lab at the moment, and
gained an understanding of their projects.
I also learned that in real-life research there can be a lot of setbacks and frustrations.
Research is not predictable and takes much longer than is immediately apparent. This project
came with a lot of challenges and it taught me how to appropriately deal with these setbacks
and come up with a new plan. I gained a lot of independence over these 8 weeks and feel
privileged to be given this opportunity.
References:
[1]
N. M. Kad and B. Van Houten, Mechanisms of DNA Repair, vol. 110. Elsevier, 2012.
[2]
K. Luger, A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond, Crystal

structure of the nucleosome core particle at 2.8[thinsp]A resolution, Nature, vol. 389,
no. 6648, pp. 251260, Sep. 1997.
[3]
F. Malouin and L. E. Bryan, Molecular Biology of the Cell, no. 4th edition, 2002.
[4]
https://www.addgene.org/vector-database/2637/.
[5]
http://www.helmholtz-muenchen.de/fileadmin/PEPF/pET_vectors/pET-28ac_map.pdf,
Identifying the intracellular survival strategies of chlamydiae

Student: Laura Pokorny
Supervisor: Dr Paul Pryor, Centre for Immunology and Infection, University of York
Daily Supervision: Hayley Clissold
Background
Chlamydia trachomatis is an obligate, intracellular pathogen that can infect a broad number of organisms ranging from
humans to free-living amoebae. C. trachomatis is a common cause of urethritis and cervicitis and long term effects include
pelvic inflammatory disease (PID), ectopic pregnancy, tubal factor infertility and reactive arthritis[1]. It has a unique
intracellular developmental cycle, whereby it alternates between two distinct morphological forms: the infectious and
metabolically inactive elementary body (EB) which is taken up into a phagocytic vesicle, and the metabolically active and
replicative reticulate body (RB), which replicates by binary fission inside the bacterium-containing vacuole termed the
inclusion. These inclusions are non-fusogenic with the components of the lysosomal pathway[2] thus can establish an
intracellular niche within the cytoplasm of the host cell, and quickly divert from the phagolysosomal maturation pathway.
C. trachomatis expresses a type-III secretion system (T3SS) which enables delivery of the bacterial effector proteins
directly into the host cytoplasm to modulate host cellular function[3], including inhibition of phagolysosomal destruction
and modulation of intracellular trafficking. Thus, the T3SS is an appealing target for the identification of pathogen
virulence factors.
Aims and overview of the project
The aim of the project was to identify chlamydial effector proteins secreted by the bacteriums T3SS that are involved in
disrupting intracellular trafficking pathways, using both a targeted approach and a random approach. The targeted
approach uses the in silico prediction program, EffectiveT3 to predict proteins that are secreted by the C. trachomatis T3SS.
These predicted proteins were then cloned into bacteria via a plasmid vector for amplification. The plasmid DNA is
isolated and used to transform BHY10 yeast strains, which contains a copy of the vacuolar hydrolase carboxypeptidase-Y
(CPY) fused to invertase (CPY-inv). The random approach uses a random genomic library of C. trachomatis genes to
transform BHY10 yeast strains. If normal trafficking is perturbed, CPY-inv is missorted and thus secreted from the cell. An
overlay solution will be laid over the colonies in order to test for CPY-inv secretion. A brown precipitate will be seen in the
case of a positive result.
Description of work
A random genomic library extracted from C. trachomatis EBs was inserted into pVT100-U vectors prior to my arrival. The
vectors were then used to transform Stellar competent bacterial cells and BHY10 yeast.
Colony PCR of transformed Stellar Competent Cells: A colony PCR was performed to amplify plasmid DNA containing the
gene of interest. 10 reactions for each gene of interest were performed (alongside a control containing 0.5l stock
undigested pVT100-U). These were then run on an agarose gel and imaged using the GeneSnap programme to check for
inserts.
MiniPrep: The plasmid from one colony that had been identified to contain the gene of interest was purified using the
protocol provided by the QIAprep Spin Miniprep Kit[4]. Due to the low copy number of the plasmid, buffer concentrations
were doubled.
Transformations: 50l Stellar Competent Cells were transformed with 5ng of DNA, 400l of SOC medium added and
plated on LB-amp plates. The yeast strain BHY10 was grown overnight in yeast extract-peptone-dextrose (YPD) broth at
30C, 250rpm. Yeast were pelleted by centrifugation at 3.3 x g for 1 minute, mixed with 100g of salmon sperm DNA
(ssDNA) and 2.25g purified plasmid DNA. 250l of PLATE solution (10mM Tris, pH7.5, 1mM EDTA, 1M LiOAc, 50% (w/v)
PEG4000) was added prior to overnight incubation at 30C. Yeast were then pelleted by centrifugation at 3.3 x g for 1
minute, resuspended in water, plated onto SC-ura plates and incubated at 30C to allow the transformed colonies to
develop.
Assay: Transformed BHY10 were streaked onto SC-ura plates alongside positive (BHY10 VPS10 + pVT100-U) and
negative (BHY10 + empty pVT100-U) yeast controls. An overlay solution containging 0.17M sucrose, 4mM sodium acetate
buffer pH 5.5, 8M N-ethylmaleimide, 1mg/ml horseradish peroxide (HRP), 160 units glucose oxidase and 10mg/ml odianiside was combined with agar (3% w/v) and poured over plates containing transformed yeast and controls. A brown
precipitate is seen when the o-dianiside is oxidised.
In the whole library screen (the random approach), the yeast were grown covering the genome 38x. 18 brown colonies
were identified after performing the assay, suggesting an intracellular trafficking defect. These colonies were restreaked
onto new SCura plates, against positive and negative controls, in order to be reassayed the next day. In the reassay, 3 of
the 18 colonies were identified to be convincing positives. These were named positive secreting colony (PSC) 6, PSC 8 and
PSC 18. Through the targeted approach, two of the predicted effector proteins gave positive results. These were
BOUR_00381 and BOUR_00916. These 5 colonies were retested on SC-ura plates with PSC 8, BOUR_00381 and
BOUR_00916 still giving strong positive results after this second assay.
A colony of PSC 8, BOUR_00381 and BOUR_00916 was picked and spread onto separate 5-FOA plates. The 5-FOA plates
allow for selection against yeast carrying the plasmid. 5-FOA forms a product that is toxic to yeast containing the URA3
gene (which is encoded on the pVT100-U plasmid). However, as yeast naturally kick out plasmids, only those yeast that
have got eliminated their plasmid will grow on the 5-FOA plate. A colony was picked from each 5-FOA plate and, after 2
days incubation at 30C, spread onto yeast-extract-peptone (YEP) plates. These were then assayed to ensure that the
result seen previously was plasmid dependent, and not a mutation in the yeast. This would be evident by the absence of a
brown precipitate. However, in the assay on the YEP, all three showed the brown precipitate (fig. 1) suggesting that the
positive results seen were due to a mutation and not the gene inserted into the plasmid.
(a)
(b)
Fig. 1. Assay of positive colonies on YEP after growth on 5-FOA. Positive colonies were spread onto YEP plates after growth on 5FOA plates and assayed to screen for trafficking defects. All clockwise from top: (a) Positive control, BOUR_00916, BOUR_00381,
negative control; (b) Positive control, PSC 8, negative control.
The whole library screen was repeated, with the genome being covered 36x this time, and 24 potential positively secreting
colonies were identified and named PSC 19-42. Of these, 18 were shown to be still secreting in the second assay. After the
third assay, three of these (PSC 20, 24 and 26) were shown to be convincing positives (figure 2).
(a)
(b)
Fig. 2. Third screen for PSCs on SC-ura. Colonies thought to be positive were spread again onto SC-ura and screened. All clockwise
from top: (a) PSC29, PSC28, PSC27, PSC26 ; (b) PSC24, PSC23, PSC22, PSC21, PSC20, PSC19
Departure from the original protocol

We aimed to isolate the plasmid DNA in the transformed yeast of any positive colonies and propagate it in bacteria. We
would then isolate that plasmid DNA and transform yeast with it in order to reassay, as well as sequencing the plasmid to
identify the gene of interest. This would be a way to check that the yeast has not mutated and to also double check that it is
a plasmid dependant result we are seeing. Unfortunately, in the time I was in the lab we didnt find any true positive
colonies and so did not get onto this stage.
Future directions
The PSCs 20, 24 and 26 will be streaked from the 5-FOA onto the YEP once growth on the 5-FOA is seen. This will then be
assayed in order to test that the positive results are plasmid dependent. Once any effector proteins have been identified,
immunoprecipitation will be performed to identify binding partners of the effector protein and isothermal calorimetry can
be used to characterise the strength of interaction. The identified chlamydial effector protein can then be crystallised so
that the full structure can be analysed and, if possible, their biological activity determined.
Value of the studentship
Not only has the studentship been extremely enjoyable, it has also been instrumental in helping me decide what I want to
do after my undergraduate degree. I now know I would like to apply for PhDs, and having the opportunity to talk to and
work with the PhD students here has been incredibly helpful. Dr Paul Pryor and Hayley Clissold have been excellent
teachers, teaching me not only important laboratory techniques but also how to methodically address unexpected issues
that may arise something that cannot be properly addressed during large, time-constrained undergraduate lab session.
By the end of the 8 weeks I was truly invested into the project and will undoubtedly keep in touch to find out where it
goes. My confidence in the laboratory has developed dramatically and I am now thoroughly excited and more prepared for
undertaking my final year project.
References
1. Paavonen J & Eggert-Kruse W. (1999) Chlamydia trachomatis: impact on human reproduction, Hum Reprod Update. SepOct;5(5):433-47.
2. Fields KA, Hackstadt T. (2002) The chlamydial inclusion: escape from the endocytic pathway. Annu Rev Cell Dev Bio.
United States. 18:221-45,
3. Betts-Hampikian HJ, Fields KA. (2010) The Chlamydial Type III Secretion Mechanism: Revealing Cracks in a Tough
Nut. Frontiers in Microbiology.1:114.
4. QIAGEN (2012) QIAprep Miniprep Handbook (PDF), p19, [Accessed: 28th July 2015]
Student : Leanne OSullivan, Biomedical Science Year 3, University College Cork

Supervisor : Dr. Paul Young, Dept. of Biochemistry, University College Cork
Construction of a novel bacteria-based DNA detection system as a simple,
low-cost alternative to current molecular diagnostics tools.
Background
Real time PCR and Quantitative PCR both currently play major roles in diagnostic labs in the detection and amplification
of target DNA sequences. These may be targets found in pathogens such as viruses or mutated genes such as in cancer
patients. The advantages of these methods is that they are quick, accurate and offer a detailed diagnosis unrivaled by
other tests. Molecular analysis makes it possible to distinguish viral and bacterial strains and also identify specific gene
mutations involved in disease, which then allows patients to receive more targeted treatments. However, these methods
of analysis are not universally available to patients. This is due to the costly nature of instruments and reagents, the need
for specialised training of staff and also the need for strict controls in procedure to prevent contamination between
samples. Where PCR diagnostics are not feasible, such as in under-resourced labs, temporary medical facilities and POC
tests in the GP office, an alternative is needed.
Aims
The aim of this project was to optimise and evaluate a novel diagnostic tool constructed by UCC iGEM Team 2014. The
system works on the principle that a DNA plasmid may only enter a competent bacterial cell and be transcribed if it is
circular. Hence by creating a linear detector plasmid that may only be circularised by binding to a specific nucleic acid
target, transformation with competent cells and transcription of a reporter gene acts as a simple readout. Binding of target
to detector is allowed by the presence of single stranded overhangs on the detector part complementary to the target. The
novel system constructed has been titled Basehunter by Cork iGEM team 2015. Over the summer, the aim of this project
was to optimise the construction protocol, assess selectivity of the system and construct multiple detectors to demonstrate
Basehunters customisability.
Methods
Results obtained by UCC iGEM 2014 suggest that construction of the detector plasmid by digestion with four enzymes
(NtbsI,NtBsQI, KpnI & HindIII) was not optimal due to presence of false positive colonies on negative control tests. It was
suspected and confirmed by sequencing of false positive colonies that this was due to undigested detector plasmid. To
overcome this, construction of the detector was attempted by PCR this summer.The detector was amplified using plasmid
pSB1C3 with cloned detector part containing the four restriction enzyme sites and the target sequence. Primers were
designed containing the target DNA sequence of the target size and annealed between nicking enzyme sites. A protocol
was designed involving amplification of the linearised (by KpnI) detector plasmid and subsequent digestion by the four
restriction enzymes. A decoy reaction was also carried out by reacting an excess of oligos complementary to the top
strand of the detector sequence with the digested plasmid to remove the top strand, yielding the single stranded
overhangs of the detector part. PCR cleanups were carried out to remove the decoy oligos and restriction enzymes and
produce the final activated detector.
Another aspect of the work carried out was the assessment of specificity of the detector system. Specificity is the ability of
the detector to select the correct target when non-target DNA is present, preventing false positives. The detectors ability
to select the correct size target was initially tested. This was done by reacting a detector for a 30bp region of HPV L1
gene with targets of sizes ranging from 30bp to 9bp.
Also assessed was the detector ability to select the target in the presence of mutated targets. These mutations included
random sequences of correct target length, 3bp changes in correct sequence and insertions and deletions in correct
sequence. These mutated and varying length targets were synthesised commercially.
Finally to assess selectivity, the sry detector (targeting a region of the sry gene on the Y chromosome) was reacted with
genomic DNA samples to evaluate whether it could differentiate male (Y chromosome) and female (no Y chromosome)
genomic DNA. To carry this out, genomic DNA was obtained from mouse tails and digested with various restriction
enzymes to release fragment of correct size for detection. Another aspect of the work carried out was the design and
construction of detectors targeting various sequences. Constructed during the project were two HPV detectors (targeting
55bp and 30bp regions of L1 gene on Human Papillomavirus), two sry detectors (targeting 62bp and 32bp regions of sry
gene) and one mycobacterium detector (24bp region of ML0319 lipoprotein gene). These were designed using sequence
analysis software, Benchling and constructed by PCR method.
Results
It was deduced that construction of the detector was optimally done using PCR as background CFUs obtained was
reduced. A decoy reaction was included in the protocol and maximised CFU achieved by positive reactions.
The selectivity of the detector was assessed and gave mixed results. Reaction with targets of various size suggested that
the detector was able to select the correct size target significantly better than smaller targets and did not detect
sequences <50% target size. However it was less efficient in selecting correct target size when larger targets were
present. Sequences which had additional bases attached to target sequence were detected almost as frequently as target
of correct size. This suggests that background colonies may be obtained where a lot of DNA is present in sample but also
that detector may react with target even if not digested to correct length. Reaction of the detector with mutated targets
revealed that the detector was highly efficient in selecting correct target where a random sequence of correct length was
present. However, where minor base changes or deletions were present, a significant number of positive CFUs were
obtained, suggesting efficiency in detecting mutations was poor.
Experiments conducted using the sry detector and genomic DNA were inconclusive. Results obtained using the digested
detector suggest that the male sample returned the most positive CFUs. However when using the PCR constructed
detector, the female sample returned the most colonies. The detector size was then reduced from 62bp to 32bp in an
attempt to improve this result, however reaction of this detector with genomic DNA was not completed in time.
Results generated using various detectors were similar and comparable, suggesting all detectors worked equally well.
Design was simple and quick, highlighting a clear advantage of the Basehunter system.
Conclusion
Overall, results suggest that the Basehunter system shows promise as a viable diagnostic tool but further work is needed
for the optimisation of selectivity of the system. Construction has been optimised to maximise performance. Selectivity
assessment suggests that the detector may be used to identify presence or absence of a target sequence, as it
successfully selects target of correct length. However, the system is currently not capable of detecting minor mutations in
sequence and further work is needed to allow this function. Also, further work is needed to optimise ability to use detector
with genomic samples as this is ultimately where the detector will be of use. In the future, these faults may be corrected.
Also, the system may be improved further by the development of a software that could locate target sequences and
design detector part and primers for amplification automatically. In addition, protocol for detector reaction may be
simplified by automating reaction and transformation. Also, the readout system may be altered to improve specificity such
as by adding a reporter gene to detector part that may be expressed by detection of correct target sequence only.
Reflection
In carrying out the summer studentship, I gained valuable experience in the world of research.
It was a great opportunity to get insight especially into the development of diagnostic tools used
in my field, Biomedical science. I became highly interested in the standards and requirements
needed for the introduction of such a system into diagnostic use and would be interested in the
future in being a part of the research and development of diagnostic tools. I learned a lot about
recording and presenting results, especially while working with others in the lab.
Communication of ideas and findings was an important aspect of everyday work that I think I
improved upon. I also got the opportunity to travel to the iGEM Giant Jamboree to present
results with the team. I met many others enthusiastic about synthetic biology there and saw
limitless possibilities in that field.
References
UCC iGEM Team 2014, Wiki : http://2014.igem.org/Team:UCC_Ireland (2014).
Cork iGEM Team 2015,Wiki : http://2015.igem.org/Team:Cork_Ireland (2015)
Sails AD (2009). "Applications in Clinical Microbiology". Real-Time PCR: Current Technology and Applications.
Caister Academic Press. ISBN 978-1-904455-39-4.
Niemz, A., Ferguson, T. M. & Boyle, D. S. Point-of-care nucleic acid testing for infectious diseases. Trends
Biotechnol. 29, 240250 (2011).
Biochemical Society Summer Vacation Studentship Report 2015

Student:
Lee Ross Burdon
Supervisor:
Dr Ritchie Williamson
Hyperthermia Induced Hyperphosphorylation of Tau in Alzheimers Disease

Alzheimers disease is a form of neurodegeneration that currently affects over 800,000 people in the
UK and is the leading form of dementia. Two distinct features are extracellular senile neuritic plaques
and intracellular neurofibrillary tangles (NFT). NFTs are composed of the protein tau which has been
shown to be hyperphosphorylated causing a decrease in taus ability to stabilise microtubules. This
leads to a loss of cytoskeleton integrity and affects the normal functioning of the cell. In addition,
cognitive decline correlates with the number of NFTs and therefore identifying the kinases responsible
for specific phosphorylation events would help researchers to develop methods to inhibit the formation
of NFTs. It has been previously reported that hyperthermia can induce changes in the
phosphorylation of tau similar to that found in Alzheimer's. The aims of the project were (I) to
determine specific sites in tau that are phosphorylated in response to temperature change (a novel
model of tau hyperphosphorylation) using phosphor-specific antibodies and phosphomapping , and
establish which of those overlap with those found in tangle pathology. (II) To identify which kinases
and phosphatases are responsible for the specific phosphorylation events in tau using the novel slices
model.
In order to meet the objectives of the study, hippocampal brain slices were obtained using a McIlwain
tissue chopper and stabilised at room temperature in oxygenated artificial cerebrospinal fluid (ACSF).
After stabilisation, slices were incubated at two different temperatures (35C or 37C) in ACSF in
order to determine changes in phosphorylation of tau dependent on temperature. Glucose use in the
brain decreases as we age and is exacerbated in Alzheimer's and so the temperature-dependent
changes in tau phosphorylation were also investigated using ACSF with different glucose
concentrations (2 mM and 10 mM). The reversible nature of tau phosphorylation was also
investigated by incubating slices at 35C for 1 hr followed by incubation at 37C for a further 1 hr and
vice versa. After incubation, slices were collected and processed for downstream analysis of
phosphorylation changes. Brain slices were homogenised in lysis buffer followed by centrifugation at
4C for 20 minutes at 15,000 RPM. The supernatant was removed and protein concentration of lysate
was determined using the Bradford assay. For analysis of tau protein phosphorylation changes, SDSPAGE followed by immunoblotting was performed with a panel of total and phospho-specific
antibodies (see Figure 1).
Tau phosphorylation appears to increase when slices are incubated at 35C compared to 37C. Total
tau antibody detected a shift in the electrophoretic mobility of tau (indicative of increased
phosphorylation) when slices were incubated at the lower temperature. This shift was reversed when
slices were switched back to 37C. In addition, tau phosphorylation was increased at the phospho
396 epitope (a site that displays increased phosphorylation in Alzheimer brain). This was more
obvious for the slices incubated in 10 mM glucose containing ACSF, a change that was not reflected
in total tubulin levels indicating a specific phosphorylation change. Further analysis revealed no
change in glycogen synthase kinase 3 (GSK3) phosphorylation at its regulatory sites (Y216 and S9).
The results indicate that hyperthermia leads to increased phosphorylation of tau but reverting back to
normal body temperate (37C) can reverse this phosphorylation providing the tissue has adequate
glucose available.
Figure 1. Representative Western blots performed using various antibodies. Hippocampal slices were
incubated in ACSF containing either 2 mM or 10 mM glucose at the indicated temperatures (C) for 1
hour, or 1 hour followed by a different temperature for a further 1 hour.
We originally intended to identify which kinases and phosphatases are responsible for the specific
phosphorylation events in tau. Unfortunately, there was insufficient time for this to be accomplished as
the viability of the slices was initially difficult to maintain. The next step is to identify further
phosphorylation changes in tau before trying to identify which kinases are responsible.
From the student:
The summer project has enabled me to develop confidence at using a variety of common laboratory
techniques which will be invaluable in enabling me to perform well in my final year project, future
research and job applications. It also made me consider my opinions on the ethics of animal research.
I am still unable to condone any form of testing on animals that causes unwarranted suffering but I
now accept that not all animal testing is beyond me. The mice were well looked after, with the ability
to exercise on a wheel, and were euthanized quickly and with minimum suffering. I would be happy to
continue performing this type of research on animals as the benefits of the research were to improve
our understanding of human disease. As for my career aspirations, I now know that I definitely enjoy
working within a laboratory but I am still unsure of the direction I will take. I would consider
undertaking a PhD, although, I would prefer to gain some industry experience first and make that
decision in the future.
I would like to say thank you to the Biomedical Society and Dr Ritchie Williamson for giving me this
fantastic opportunity and also to everyone else that gave me help and advice throughout the project.
From the supervisor:
The benefits of this project were two-fold, it provided an excellent opportunity to encourage talented
undergraduate students to pursue a career in basic research (especially in neurodegeneration), and
to give real experience of research as opposed to the 'cook book projects' they undertake as part of
their undergraduate program. It also validated our laboratories approach in trying to model complex
neurodegenerative correlates without recourse to whole animal in vitro studies.
Background
Complement is a vital branch of the innate immune response and consists of numerous plasma proteins which have a
collaborative role in host-defence against foreign pathogens. The complement system can be activated by three activation
pathways: the classical, lectin and alternative pathways which congregate at C3, which is the central protein of the complement
system. Unlike the classical and lectin pathways, the alternative pathway is constitutively active; this tick-over mechanism
allows the system to be primed for full activation. The alternative pathway is initially activated via the spontaneous hydrolysis of
a thioester bond in C3; this initiates a cascade resulting in the formation of a C3 convertase called C3bBb. This convertase results
in a positive feedback amplification loop by cleaving more C3 into immunologically active C3b.
The effector functions of complement are extremely potent and have the potential to harm the host. Hence, the downregulation of these functions on host cells, whilst not detrimentally impacting their effect on foreign pathogens, is crucial. A
major complement regulatory protein is Factor H; it has a significant contribution to the protection of host cells. Factor H has
multiple roles; these include: binding C3b, C3bBb decay acceleration and cofactor activity for Factor I mediated C3b proteolytic
inactivation. C3b is degraded to iC3b and eventually to C3c and C3d. Three C3b binding sites on Factor H have currently been
identified and these are located on short complement regulator domains 1/4, 6/8 and 19/20 (1). As C3b and C3u are functionally
synonymous, structural studies carried out on C3b-FH complex formation are analogous to those with the C3u-FH complex
formation. Hydrolysis of the thioester domain in C3 produces C3u, whereas C3b is generated via C3 cleavage to remove C3a.
Recently, conflicting data has emerged which raises uncertainty of the previously assumed 1:1 binding valency between Factor H
and C3b; with studies suggesting a bivalent interaction with two C3b molecules binding at opposite ends of Factor H. (2;3)
Aims
Perform analytical ultracentrifugation studies in order to elucidate solution structures of the complex formed between Factor H
and C3u to determine whether this has a 1:1 or 2:1 binding stoichiometry under physiological conditions.
Experimental Procedures
Purification of Complement Factor H CFH was purified in micromolar amounts using five chromatographic stages via an inhouse protocol. CFH was purified from frozen human plasma stock; the plasma was thawed and centrifuged to remove
precipitated material. The supernatant was collected, filtered and then dialysed in running buffer. Subsequently, the
supernatant was passed through a gravity-driven non-immune immobilised IgG on Sepharose column. This was followed by a
Lysine-Sepharose column and then an immunoaffinity column using MRC-OX23 anti-FH monoclonal antibody; the CFH was
eluted from the column using 3M MgCl2 solution. Next, the CFH was repeatedly dialysed in HEPES buffer and then passed
through a GE HiTrap Protein G HP column to remove antibody contaminants. Finally, the CFH solution was concentrated in a
centrifuge using centrifugal filter units and gel filtrated using a GE Superose 6 column to remove any other contaminants.
Purification of C3 Initially, EDTA was added to freshly collected venous blood in order to prevent clotting. The blood was then
spun down to separate the plasma from the other blood components. The plasma was centrifuged further and PEG was added
to promote precipitation; helping to increase the relative concentration of C3 in the plasma. Pefabloc was then added to
prevent proteolytic degradation before purification and the plasma was manually filtered using a filter unit. The first
chromatographic stage involved using a GE Q-Sepharose anion exchange column run with an KTA purification system, C3 was
eluted using a NaCl gradient. The second column used to purify C3 was a GE MonoQ anion exchange column; this removed
major contaminants in the plasma and C3 was again eluted with a NaCl gradient. The final purification step, which took place
after the C3 had been centrifugally concentrated, was gel filtration using a GE Superose 6 column. This step removed any
degradation products and other minor contaminants that may have been present. The purified C3 was then hydrolysed to C3u
by incubation with hydrazine. Finally a functional assay was carried out to confirm the C3 to C3u conversion using the
complement regulators CFH and CFI, which cause proteolytic degradation of C3u, but not C3.
Analytical Ultracentrifugation Analytical ultracentrifugation (AUC) is a versatile technique which allows quantitative analysis of
macromolecules in solution. Sedimentation data is logged by optical systems which observe samples during sedimentation
under a sufficient gravitational field. In recent experiments, AUC has shown the presence of the 2:1 complex; however, this
deduction was inconclusive as the concentrations of protein used were too low. After purification, various mixtures and
concentrations of CFH, C3, and C3u were sedimented to study the stoichiometry; the data was analysed using SEDFIT software.
Assay of cofactor activity for
Factor I-catalysed cleavage of
C3u. The inferred identities of
Coomassie-stained protein bands
on an SDS-PAGE gel are shown.
The functional assay was carried
out with excess FI and C3u in a 2:1
ratio with FH in micromolar
amounts.
Complement Factor H and C3u were used for two AUC analyses. The initial experiment was carried out using a 1:1 ratio of
o
o
o
C3u:FH at 6.2 M and the second experiment at 9 M. The AUC analyses were performed at 6 C, 20 C and 30 C and the data
collected were interpreted using a SEDFIT analysis. The first experiment resulted in three peaks which corresponded to FH, C3u
and a 1:1 complex. The binding complex peak was relatively low in comparison to peaks suggested by other studies; this factor
in addition to the lack of a 2:1 peak gave cause for further investigation in a second experiment at higher concentrations. Results
from the second analysis were very similar and peak integration allowed the K d values to be calculated. The Kd values were 29.05
o
o
o
M and 30.37 M for the first experiment at 6 C and 20 C respectively and 78.46 M for the second experiment at 20 C.
The overall results collected show a reproducible formation of a weak transient complex as indicated by the relatively high Kd
values. The lack of a 2:1 binding complex between FH and C3u suggests the non-existence of this form of complex, this may be
due to the complexes being mutually exclusive of one another which allows only a 1:1 complex to form.
Departures from Original Proposal & Future Directions
There were no deviations from the original proposal. The data will be used to supplement earlier studies of this complex with a
view to its eventual publication.
Value of Summer Studentship
This studentship has been instrumental in compounding my desire to pursue a research laboratory based PhD. Moreover, the
opportunity to develop my scientific writing, communication and presentation skills has substantially increased my confidence
levels, especially when interacting with colleagues of all ages in a work environment. My practical competence has improved to
a point where I actively search for occasions to try out new techniques and equipment. I have not only gained a deeper
understanding of life in the laboratory, but also how to deal with problems which arise from all possible aspects of a professional
setting. I have learnt to work collaboratively with other members of the group in addition to working independently without
needing assistance or reassurance. Accordingly, I am now looking forward to working on my final year project rather than being
subjected to my previous apprehension. I have truly appreciated being part of a project with significant scientific relevance and
also being provided with the opportunity to use and be trusted with niche technology which I havent experienced as part of my
degree course. Professor S. J. Perkins adds: The summer project by Louis Buckley progressed very well and we are more
confident of our understanding of the stoichiometry of the C3u-CFH complex. We hope to publish our conclusions in due course.
References
1.
Jokiranta TS, Hellwage JF, Koistinen VF, Zipfel PF FAU, Meri S. Each of the three binding sites on complement factor H interacts with a
distinct site on C3b.(0021-9258 (Print)).
2.
Kajander T, Lehtinen MJ FAU - Hyvarinen S, Hyvarinen S FAU - Bhattacharjee A, Bhattacharjee AF, Leung E FAU - Isenman D, Isenman DE
FAU - Meri S, et al. Dual interaction of factor H with C3d and glycosaminoglycans in host-non host discrimination by complement.(10916490 (Electronic)).
3.
Wu J, Wu YQ FAU - Ricklin D, Ricklin DF, Janssen BJ FAU - Lambris J, Lambris JD FAU - Gros P, Gros P. Structure of complement fragment
C3b-factor H and implications for host protection by complement regulators.(1529-2916 (Electronic)).
RNA processing in the Plasmodium falciparum apicoplast

Louis Wilson
Department of Biochemistry, University of Cambridge
Supervisors: Dr Ellen Nisbet, Prof. Chris Howe
Rpl2 protein sequence analysis was also conducted using BLAST, while
structural analysis involved the use of the open-source molecular
visualisation system PyMOL.
Results
Introduction and Project Aims
Apicomplexan parasites have become remarkable in recent
years for the discovery of their algal ancestry. Most
apicomplexans, including Plasmodium spp., possess a
remnant chloroplast the apicoplast complete with its
own genome and proteome (albeit small in size). Since
antimalarial drugs such as doxycyline and clindamycin target
apicoplast gene expression, it is in the interest of researchers
to understand more clearly how apicoplastic genes are
expressed continued research may lead to the
development of further drugs.
In Plasmodium falciparum, it is currently understood that
genes in the apicoplast are initially expressed as a small
number of long, polycistronic transcripts, which, in order to
be translated, are subsequently cleaved into individual open
reading frames (ORFs) by RNA processing enzymes. It has
also more recently come to light that the ribosomal proteinencoding gene rpl2 undergoes a GA editing event towards
the 3 end (Nisbet et al 2015, submitted). The aim of this
project was to observe snapshots of RNA intermediates at
different stages of such processing, and determine the point
at which RNAs are edited, as well as examining the general
processing mechanism (an addition to the original proposal)
using the procedure below. A bioinformatic analysis was also
to be conducted in order to identify potential reasons for
editing to be necessitated in vivo.
Experimental methods
Prior to the project, parasites were subjected to a treatment of rifampicin,
an inhibitor of RNA polymerase, by Dr Nisbet for either 1, 2 or 6 hours
before initiating the first step of RNA extraction. During the project, RNA
extraction was completed; RNA was then circularised using an RNA ligase.
First strand synthesis employed a primer complementary to a site near the
3 end of rpl2, creating a cDNA concatemer (polymerisation continues
several times around the circle). Reverse transcriptase was not added to a
second set of similarly-treated RNA preparations, which were retained as
negative controls.
Obtaining the desired cDNAs with minimal contamination

proved to be very difficult. This may have been due to RNA
samples being of insufficient quality or quantity (rifampicintreated cells intuitively contain less RNA than is typically
encountered), but was at least partly caused by exogenous
DNA contamination (or failure to fully digest DNA during the
extraction procedure). However, one cDNA preparation
appeared successful following diagnostic electrophoresis
(see Figure 1), and provided the bulk of the data collected
during the project.
Unfortunately, due to unanticipated deterioration of the
ampicillin stock, three quarters of the cells transformed with
the PCR products originating from this cDNA had to be
discarded due to bacterial contamination. Nevertheless,
several colony PCRs were conducted to analyse the many
remaining transformants. A large proportion of identified
transformants proved to possess only a very small insert in
the plasmid vector (a few tens of base pairs), which was
demonstrated by the negligible difference in band size
between these clones and blue colonies assayed as negative
controls.
Initially, efforts were primarily focused on purifying plasmids
containing the larger inserts, which should yield the
sequences of interest. A list of implied RNA species was then
generated by analysing the derived sequences in silico (see
Table 1). Most of the transcripts sequenced had already
been edited by the time RNA was extracted; this suggests
that the editing occurs quite early relative to mRNA cleavage
events, although more data are required to confirm this
conclusion owing to the surprising existence of unedited
transcripts from the 6-hour treatment. In addition, the assay
Outward-facing primers annealing to sequences at each end of rpl2 were

then used to amplify the intervening sequence containing the regions
adjacent to the gene by PCR (their termini ligated during the circularisation
reaction). PCR using conventional inward-facing primers was also
conducted as a control.
PCR products were examined using gel electrophoresis, before cloning into
a plasmid vector. Competent Escherichia coli cells were transformed with
the plasmid by heat shock transformation, and were spread on ampicillin
plates to permit blue-white selection of positive transformants. White
colonies were checked for successful transformation by colony PCR
(primers annealing to sequences either side of the insert) before culturing
and mini-prepping. Purified plasmids were submitted to an internal facility
for sequencing, and the sequences themselves analysed using NCBIs BLAST
algorithm.
Fig. 1. PCR amplification of cDNAs synthesised from

circularised rpl2 transcripts extracted after 1, 2 or 6 hour
treatment with rifampicin. Internal primers permitted
conventional amplification of the rpl2 locus, whereas
circular primers amplified the encompassing sequences.
100 kb DNA ladder was added to the first and fourteenth
wells.
Table 1. The 5 and 3 ends of cloned apicoplast rpl2 mRNA

molecules were mapped to positions in the genomic sequence.
Due to primer locations, the assay lacks the power to detect
transcripts cleaved between positions 2886 and 3453 (this region
is contained by the rpl2 ORF).
Treatment
5 end
3 end
Length
Edited?
2824*
3916*
1092
Yes
2392*
3738*
1346
Yes
2750*
4202*
1452
Yes
1909*
3961*
2052
Yes
1 hr
2857
3846
989
Yes
1909
3846
1937
Yes
2773
3564
791
Yes
?
3739
N/A
Yes
2 hr
2506
3876
1370
Yes
2869
4493
1624
Yes
2756
4026
1270
No
2785
4103
1318
Yes
2873
4494
1621
Yes
6 hr
2873
4494
1621
Yes
2756
4026
1270
Yes
2785
4103
1318
Yes
2756
4026
1270
No
* 1; 3
lacks the capacity to detect editing in unprocessed

transcripts, as they possess multiple phosphate residues at
the 5 end that prevent their circularisation.
Examining the points at which the transcripts are cleaved
provided some interesting observations. Several transcripts
shared the same position of cleavage at one end (but not
the other), advocating a degree of sequence-specificity in
endonuclease activity. Many of the sites also corresponded
to the precise 5 or 3 ends of ORFs. 3 processing did not
appear to be co-ordinated with 5 processing.
reported to be of large importance to the peptidyl

transferase activity of the ribosome (Diedrich et al., 2000).
Future directions
Clearly, more data are required to strengthen the findings of
this project. Ideally, new cultures should be grown to
provide fresh RNA samples, and systematic replacement of
reagents might be warranted to eliminate DNA
contamination. It might well also be prudent to repeat the
experiment using different primers, especially given the
proximity of current primers to the site of editing itself.
An interesting subject for further investigation might be the
enigmatic antisense mRNAs that appear to exist for every
apicoplastic transcript. The processing of these transcripts
could be analysed to determine whether they are processed
and edited in the same fashion as conventional mRNAs.
Value of studentship to student
I have learnt a great deal from this studentship. This project
has provided a unique opportunity to experience science in
practice to see how research is actually carried out in the
laboratory. Owing to the difficulty in conducting the above
experiments successfully, I spent a large amount of time
working out why assays were not working this, in itself, is a
critical skill in research, and I am glad to have had to
experience it. In addition to becoming more aware of the
practical aspects of research, I have had the chance to
practise many biochemical techniques (including some
protein work on the side of the project) that will put me in
good stead for the remaining years of my degree and,
hopefully, a PhD. This studentship has strongly reinforced
my aspiration to become an academic researcher.
Value of studentship to laboratory
Contaminating sequences were also detected; while some of

these were derived from other taxa, a large proportion of
cloned contaminants were primer dimers of sorts. Many of
these primer dimers contained small inserts of 315 bp
length, and were often of indeterminable origin. However,
one such insert, of sequence ATGACCCCT, was consistently
detected across several preparations, albeit sometimes
truncated. Since the first four bases are identical to those
following the forward primer in an edited transcript (the
fourth being the edited base itself), and the next five bases
are complementary to the site of editing in an unedited
transcript, it remains to be seen whether this is a by-product
of the editing mechanism itself, or simply an artefact of the
proximity of reverse transcriptase priming.
The G650A edit in rpl2 results in a codon change from
glycine to glutamate at position 217 in the encoded protein,
ribosomal protein L2 (Rpl2). By comparing the primary
sequence of Rpl2 with those of orthologues in a wide range
of taxa, it was found that the glycine edited in the P.
falciparum apicoplast is highly conserved. Therefore, the
edit does not correct an aberrant residue to one that is
conserved, contrary to the previous hypothesis. Structural
analysis of the Rpl2 orthologue in E. coli indicated that the
comparable residue, Gly-232, was very close in space to a
pair of residues, Asp-228 and His-229, that have been
We greatly enjoyed hosting Louis in the laboratory over the

summer. He worked extremely hard and was able to
produce some excellent data. These have confirmed our
suspicion that RNA editing in the Plasmodium apicoplast
occurs early, and is important. We hope to include his data
in a future publication, and also in future grant applications
for further analysis of apicoplast transcription.
Dr Ellen Nisbet, 1 Oct 2015
References
Diedrich, G., Spahn, C. M., Stelzl, U., Schfer, M. A., Wooten, T., Bochkariov,
D. E., Nierhaus, K. H. (2000). The EMBO Journal, 19, 524150.
Interaction of Parkinsons and Lysosomal Related Genes

Background and Aims
Parkinsons disease (PD) is primarily associated with movement disorders. However patients also report nonmotor symptoms including a range of visual defects such as dry eyes, double vision and hallucinations. The
Lrrk2-G2019S mutation is the most common form of genetic PD. Both fruit flies and mice are commonly used
to model PD. Mice models, however, fail to faithfully recapitulate phenotypes associated with visual
dysfunction, fly models conversely have been shown to produce reliable PD phenotypes associated with visual
system. Previous work has shown that expression of Lrrk2-G2019S in dopaminergic neurones results in an
initial gain in visual response in young flies followed by a decay consequently leading to compromised visual
(1)
(2-4)
function . The G2019S mutation has also been linked to disruptions to the endolysomal system
. Evidence
(2, 4)
(3, 5)
suggests the function of Lrrk2 occurs in a Rab dependant manner; with both Rab7
and Rab7L1
(drosophila orthologue Lightoid) implicated as potential interaction partners.
Using the visual system of model organism Drosophila melanogaster, we aimed to probe for genetic
interactions between Rab7 and Rab7L1 with Lrrk2-G2019S; to provide novel evidence for the function of Lrrk2G2019S and its role in the pathogenesis of PD.
Work Carried Out
Expression of Rab7 or Lightoid was upregulated by constitutive expression or knocked down by RNAi using the
GAL4/UAS system. Transgene expression was driven specifically in the dopaminergic neurones using Tyrosine
hydroxylase-Gal4 (TH-Gal4) and interactions were tested for with G2019S. Interactions were also tested for
apr
between G2019S and w (negative control) or -Synuclein-30P (positive control). The visual response of the
transgenic flies was tested at 1 and 7 days using the SSVEP (steady state evoked visual potential) technique.
Whereby, flies were restrained in a Gilson pipette tip and exposed to full field illumination by a blue LED driven
by a flickering wave. The response of the visual network was then recorded by electrodes, one placed on the
eye and the other the mouth.
Results
Our results (Fig 1A, 1B) showed several things of note. Firstly, the results clearly show that the function of both
Rab7 and Lightoid in dopaminergic neurones is linked to the visual response; RNAi of either gene resulting in a
large gain in visual response. Moreover, Lightoid did not appear to interact with Lrrk2. The visual response of
Lightoid flies not differing significantly between flies expressing only the Lightoid transgenes or the transgenes
in the background Lrrk2-G2019S, when compared to the negative control flies. Conversely a clear interaction
was observed with Rab7. Flies constitutive expressing Rab7 in the G2019S background showed a significant
increase in visual response compared to those only expressing Rab7 alone. A similar but less pronounced result
was observed in the Rab7 RNAi flies. These phenotypic differences were visible in 1 day old flies but became
more pronounced by 7 days. By testing at both one and seven days we ensured small phenotypes, which did
not differ significantly from the controls at one day, yet increased overtime would not be dismissed, giving
more confidence in our conclusions. The -Synuclein-30P mutant (positive control) has been linked to
inherited PD. Flies expressing -Synuclein-30P showed a clear phenotype with a significant increase in
response when in the G2019S background, further reinforcing our conclusions. Surprisingly, our results did not
show a clear phenotype for G2019S alone. It is possible that this is a consequence of the genetic background
apr
of the fly eyes studied; here flies with a w eye were used, whereas the G2019S phenotype was reported in
flies with a w eye. These results therefore support an interaction with Lrrk2 and Rab7 not Rab7L1, however
caution should be taken with the interpretation of data from recombinant animal studies and further work is
needed to validate this result.
Future directions
The G2019S mutation is characterised by increased kinase activity, to validate our results the transgenes used
here should be expressed with Lrrk2-G2019S and targeted kinase inhibition. Previous work showed the visual
response of Lrrk2-G2019S decayed over extended periods of time or if flies are repeatedly exposed to flashing
light and future work should look to see if this phenotype is replicated by the genotypes studied here.
1A
1B
apr
Fig. 1. Rab7 but not Rab7L1 interacts with Lrrk2-G2019S. Visual response of flies measured using SSVEP. W
(negative control) and -Syn-30P (positive control). Error bars showing one standard error of the mean. At least n = 9
flies of each genotype were tested at 1 (Fig. 1A) and 7 (Fig. 1B) days of age.
Departures from the original proposal

It was originally intended that as a second objective the HRS and ALIX genes which function in lysosomal
trafficking by the ESCRT complex would be examined to provide evidence for a secreted role of Lrrk2. This was
abandoned and instead Lightoid was manipulated alongside Rab7 to test current hypotheses for the
interaction partners of Lrrk2. It was also intended to corroborate results from a physiological reporter with an
anatomical reporter however stock contamination of flies used for the later rendered this unfeasible in the
time frame.
From the Supervisor
Martin worked hard on this project, and collected an outstanding amount of good data. He learnt a great deal
about time management, record keeping and data analysis as well as a deep understanding of the roles of
Rabs and Lrrk2. He was able to test published hypotheses about the roles of Rab7 and Rab7L1 in the fly model
of Lrrk2-G2019S induced PD. He clearly showed that Rab7L1 interacts with Lrrk2-G2019S in a different way to
Rab7. We have already begun follow up experiments to confirm and extend his conclusions, as it is essential to
try and understand the interactions of Lrrk2-G2019S. We will study the effects of other Lrrk2 mutations
associated with PD on these two Rabs, the effects of synthetic, kinase dead, mutations, and of a protective
Lrrk2 variant. We will revisit the developmental phenotype using a cleaner GMR-GAL4 line.
The success of this project opens a new area of work for the lab. Once Martins conclusions have been
followed up in this way, we will be in a position to extend them to other Rabs with grant support.
From the Student
This grant allowed me the opportunity to undertake a summer studentship. Through which I have gained
valuable experience of working a laboratory and confirmed my interest in research and my decision to pursue
further study.
References
1.
Afsari F, Christensen KV, Smith GP, Hentzer M, Nippe OM, Elliott CJ, et al. Abnormal visual gain control in a Parkinson's disease
model. Human molecular genetics. 2014;23:4465-78.
2.
Dodson MW, Zhang T, Jiang C, Chen S, Guo M. Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal
positioning. Human molecular genetics. 2012;21:1350-63.
3.
MacLeod DA, Rhinn H, Kuwahara T, Zolin A, Di Paolo G, McCabe BD, et al. RAB7L1 interacts with LRRK2 to modify intraneuronal
protein sorting and Parkinsons disease risk. Neuron. 2013;77:425-39.
4.
Gmez-Suaga P, Rivero-Ros P, Fdez E, Ramrez MB, Ferrer I, Aiastui A, et al. LRRK2 delays degradative receptor trafficking by
impeding late endosomal budding through decreasing Rab7 activity. Human molecular genetics. 2014;23:6779-96.5.
Beilina
A,
Rudenko IN, Kaganovich A, Civiero L, Chau H, Kalia SK, et al. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a
common pathway for sporadic and familial Parkinson disease. Proceedings of the National Academy of Sciences. 2014;111:2626-31.
Biochemical Characterisation of Tsr3, a Novel Candidate Endoribonuclease in Yeast

Ribosome Biogenesis, and its Putative Co-factor and Chaperone Tsr4
Student: Matthew Wilkinson, Supervisor: Dr Claudia Schneider, Newcastle University
Background
Ribosome biogenesis is one of the most important tasks undertaken by any eukaryotic cell, requiring around 80% of
a growing cells energy. It is also immensely complicated, taking place in both the nucleus and the cytoplasm, and
involves over 300 assembly factors. In Saccharomyces cerevisiae (budding yeast), the 18S, 5.8S and 25S ribosomal
RNAs (rRNAs) are transcribed in the nucleus as a single long precursor molecule (pre-rRNA) that is then cleaved into
the mature rRNAs by multiple endo- and exonucleases. Nob1 is the endonuclease that cleaves the 20S pre-rRNA at
site D to generate the mature 18S rRNA and it interacts with a binding partner, Pno1. Interestingly, abolishing Nob1
activity in vivo does not completely stop 18S rRNA formation, suggesting another endonuclease must be present that
potentially works as a backup enzyme for Nob1. Tsr3 has been identified by bioinformatics (Burroughs and Aravind,
2014) as this potential backup site D endonuclease and it has been hypothesised to interact with a binding partner
Tsr4, an essential protein in yeast. The role of Tsr3 is currently unknown and there are many suggestions as to its
role. Ideas include that it directly modulates Nob1 activity, it directly cleaves the 20S pre-rRNA at site D or that it
plays a more subtle structural role in the pre-ribosomal complex.
Aims
The key aim of the project was to produce purified recombinant proteins of Tsr3, Tsr4, Nob1 and Pno1 that could be
used in protein-protein interaction studies and to carry out pre-rRNA cleavage assays. Another aim was to apply sitedirected mutagenesis to generate mutant constructs to probe the importance of residues in the hypothesised active
site of Tsr3. In an addition to the original project, it was also aimed to co-express and purify another pre-rRNA
endonuclease, Utp24, and its cofactor Utp23 in order to probe the interactions between the two through in vitro
crosslinking.
A very important aim of the studentship was also to give me an idea of what working in a busy research lab would be
like in order to inform my career decisions and to help me develop important skills such as time management,
decision making and key practical skills.
Methods
The open reading frames for the Tsr3, Nob1, Pno1 and Tsr4 proteins were amplified from yeast genomic DNA and
cloned into the pET100 vector to add an N terminal His tag to the expressed protein. Tsr3 and Nob1 were also subcloned into the pGEX vector to add an N terminal GST tag. Utp23 and Utp24 were subcloned into a single pGEX
vector so that Utp23 had a GST tag and Utp24 had a His tag. Site-directed mutagenesis was carried out on the
bacterial expression vector encoding His-tagged Tsr3 to create an Aspartate to Asparagine change at position 57
(D57N). It was attempted to mutate both GST- and His-tagged Tsr3 and Nob1 (D15N), but only the His-Tsr3 D57N
mutant was successfully created within the given time frame. All vectors were then transformed into BL21 E.coli
cells, grown up into large scale cultures and recombinant protein expression was induced with IPTG. Cultures were
then lysed and protein purifications were performed using Nickel sepharose (for His-tagged proteins) or glutathione
sepharose (for GST-tagged) proteins.
Protein-protein interaction studies (pulldowns) were undertaken, where the GST-tagged proteins were immobilised
on glutathione sepharose beads before being incubated with His-tagged proteins. The beads were washed to
remove any unbound protein and the immobilised proteins were extracted, separated by SDS PAGE and analysed by
Western blotting. Crosslinking of Utp23 and Utp24 was carried out by adding different concentrations of the
chemical crosslinker BS3 to purified samples, followed by SDS PAGE and Western blot analysis.
Results
All proteins were successfully expressed and purified including the mutant Tsr3 protein (data not shown). The D57N
mutation in Tsr3 had previously been confirmed by sequencing. Pulldown studies showed that there is a multitude of
interactions between all of the different recombinant proteins tested, which is summarised in figure 1. This can also
be seen from the western blot that was performed to analyse the pulldown experiments, an example for this is
shown in figure 2. Crosslinking was also successfully applied to Utp23 and Utp24 complexes (figure 3).
Crosslinked
proteins
Figure 1. Each arrow shows a

protein-protein interaction as
seen in pulldown experiments.
Figure 2. Each dark signal

represents an interaction
between two proteins. GST was
used as a negative control.
Figure 3. As the ratio of crosslinker

to protein increases the weight of
the band increases to show
multimer formation
Discussion
In this studentship, it has been shown that Tsr3 and Tsr4 do interact with each other as hypothesised, albeit only
weakly, and that Tsr3 is likely to be involved in ribosome biosynthesis due to its strong association with Nob1. It was
planned to carry out in vitro cleavage assays using both wild type and D57N Tsr3 on 20S pre-rRNA, however there
was not enough time to accomplish this. However, by generating the purified proteins this will greatly help the
Schneider lab to carry out these studies in the future.
Further work to build on this studentship is currently being carried out in the Schneider lab through investigating the
in vivo role of Tsr3 using S. cerevisiae. The next step in the crosslinking experiment is to optimise the process and
then use mass spectrometry to identify the individual amino acids that are interacting with each other in order to
describe the interaction surface between Utp23 and Utp24.

I found the studentship extremely enjoyable and it was valuable in giving me an idea of what doing a PhD would be
like and how research works on a day-to-day basis. This has helped me to decide that I want to study for a PhD after I
graduate. The lab experience and other valuable skills that I have gained from the studentship will be very useful not
only to my final project but to my future career and studies.
Furthermore, it was very motivating to hear from my supervisor that the data generated by myself has really kickstarted a new research project in the laboratory.
References
Burroughs AM and Aravind L (2014) Analysis of two domains with novel RNA- processing activities throws light on
the complex evolution of ribosomal RNA biogenesis. Front. Genet. 5:424
Biochemistry Society Summer Studentship Report 2015

Student: Michaela Dermendjieva
Supervisor: Prof Robin Allshire
Direct Supervisor: Dr Manu Shukla
Affiliation: Institute of Cell Biology, University of Edinburgh
Evaluation of proximity tagging approaches for identification and

analyses of CENP-A chromatin-associated factors in S. pombe
Introduction
Centromeres are chromatin structures where kinetochores assemble during cell division. Kinetochores are large
multiprotein complexes which facilitate the attachment of chromosomes to the microtubules and the proper
segregation of sister chromatids. The sites of centromere formation are defined epigenetically by the presence
of the specialized histone H3 variant CENP-A (centromere protein A). Some CENP-A-associated proteins have
been identified so far [1] but further cataloguing of such factors is needed to elucidate the mechanisms of CENPA assembly. Studying these proteins in the fission yeast S. pombe has been difficult due to technical limitations.
To overcome these limitations, we evaluated two recently-described proximity tagging approaches.
1. BioID (proximity dependent biotin identification) is a method based on a mutated prokaryotic biotin
ligase (BirA*) which is fused to a protein of interest and promiscuously biotinylates proteins in its vicinity [2]. The
biotinylated product is then isolated by streptavidin-based biotin affinity capture. An additional benefit of this
system is that transiently interacting proteins can be identified.
2. APEX is a system based on an engineered ascorbate peroxidase (APEX) which similarly to BirA*
biotinylates neighbouring and interacting proteins when biotin-phenol is present in the reaction [3, 4].
Aim
The aim of the project was to evaluate two proximity tagging approaches (APEX and BioID) in the fission yeast
Schizosaccharomyces pombe for identifying CENP-A chomatin-associated proteins.
Departures from Original Proposal

There were no major departures from the original proposal.
Description of work
To target the engineered enzymes to the centromeres, we mainly used direct fusion constructs of BirA*/APEX
with CENP-A. These constructs were introduced and expressed in S. pombe cells via plasmid transformation. In
addition, this preliminary stage included performing colony PCR to determine transformation efficiency. An
alternative indirect tagging system based on GFP-GBP interaction was used in the initial BioID experiments.
To overcome the physical barrier of the yeast cell wall, we performed partial permeabilization of the cells by
enzymatic digestion reaction. Then we isolated the insoluble chromatin fraction and performed in vitro
biotinylation to facilitate the labelling of CENP-A-proximity proteins. To assess the final product, we performed
western blotting and streptavidin-based affinity enrichment to detect the biotinylated proteins in the chromatin
fraction.
A series of experiments was aimed at optimization of the experimental conditions. Attempts to better suit the
protein extraction method for both of our BioID and APEX experiments included performing co-IPs with
streptavidin beads. Optimization of the biotinylation conditions for APEX included H2O2 titrations. To improve the
development conditions we tested for a better enrichment reagent.
Results and Outcomes

BioID experiments. We successfully managed to obtain a crude chromatin fraction of our strains and showed
that the biotinylating proteins remain in the soluble fraction while the chromatin-associated biotinylated proteins
remain in the crude chromatin fraction (Fig 1.).
1
+ Biotin, + hemin
+ Biotin, - hemin
- Biotin, + hemin
APEX strain
Control Strain
Endogenous
biotinylating
proteins
BirA*
BirA*-GBP-NLS
GFP-CENP-A
BirA*-GBP-NLS
Figure 1. In vitro biotinylation performed

on pombe strains expressing BirA*-GBPNLS construct (indirect tagging) or BirA*CENP-A construct (direct tagging). 1
supernatant, 2 crude chromatin
fraction, 3 crude chromatin fraction +
biotin. Western blot with streptavidinHRP.
Control strain
APEX-CENP-A strains
APEX-CENP-Aexperiment
strains
Figure 2. In vitro biotinylation
with APEX-CENP-A-expressing strains. Red
arrows pointing towards unique bands.
Figure 3. Co-IP protein

extraction of biotinylated
proteins after in vitro
biotinylation of crude
chromatin fraction. Silver
staining method.
APEX experiments. We could observe several unique bands of biotinylated proteins in one of our in vitro
biotinylation experiments but due to much background noise no conclusions can be made about the success of
our system (Fig. 2). However, overall increase in biotinylation could be observed in our co-IP experiment with
APEX-CENP-A expressing strain (Fig. 3).
In conclusion, some activity in the APEX system could be observed but further optimization of the development
and extraction methods could increase the efficiency of this technique in the future.
Future Directions
Further experiments can be performed with the APEX system to optimize the biotinylation conditions, for
instance the timing over which the reaction should take place can be tested for. Additionally, a further improved
version of APEX has been recently described which can be evaluated.

To the Student:
This project has been an incredible insight for me into what it is like to be involved in research. It was an
opportunity to not only learn new techniques and get hands-on experience, but also learn how to go about when
trying to verify a system and what to take into account when designing an experiment. Adapting a method
turned out to be a challenge but I learnt so much about the reasoning behind every step and the limitations of
analytical tools. I made a lot of observations about the research profession which together with my own work
made me very positive about pursuing a career in science.
To the Lab:
During her stay, the student managed to evaluate many optimization parameters towards setting up the
methodology. These results will serve as valuable groundwork for further optimizations. Additionally, the
studentship allowed a mentorship opportunity for a postdoctoral researcher in the lab, which is extremely
important for their development as future mentors.
References
1.
2.
3.
4.
Perpelescu, M. and Fukagawa, T. (2011) The ABCs of CENPs. Chromosoma, 120, 425-446
Roux, K.J., Kim, D.I., Raida, M. and Burke, B. (2012) A promiscuous biotin ligase fusion protein
identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196, 801810
Martell, J.D., et al.(2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron
microscopy. Nature Biotechnology, 30, 1143-48
Rhee, H.W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A. and Ting, A.Y. (2013) Proteomic
mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science, 339, 1328-1331
Student: Mnica Esteban Garcia

Supervisor: Dr Akane Kawamura
Summer 2015
CRL-Department of Chemistry, University of Oxford
Oxidative Stress and Epigenetics: Study of the inhibition of KDM4A by H2O2

Aims of the project:
Epigenetic regulation is the mechanism by which cell differentiation and specific cell expression is
possible. Its deregulation has been linked to many disorders, such as different types of cancer,
inflammation-related diseases or aging(1). A wide array of enzymatic families is responsible for
genomes integrity and accessibility to transcriptional machinery, by inducing modifications on
histones such as demethylation(2). JmjC-KDMs belong to a family of Fe(II)/ 2-oxoglutarate (2OG)
dependent oxygenase, that require oxygen to carry out their function. KDMs are histone
demethylases that target specific lysine residues on histones, presenting different states of
methylation. KDM4 can demethylate tri-methylated lysine residues of H3K9me3 and H3K36me3. It
can also use H3K9me2 and H3K36me2 as a secondary substrate, so it can also result in formation of
H3K9me1(3). H3K9 methylation is often associated to silenced parts of chromatin, but has also been
found in transcriptionally active genes, and vice versa applies to H3K36, so the final effect of KDMs
action seems to fall into a more complex set of interactions(4).
Detrimental epigenetic modifications have been associated with oxidative stress, resulting in
aberrant patterns of gene expression. Oxidative stress is the result of many cellular metabolic
processes, such as imbalance of cellular redox haemostasis due to excessive production of reactive
oxygen species (ROS)(5). Hydrogen peroxide is one of the naturally produced ROS in the human
body, and is neutralized to a certain extent by antioxidant. However, an imbalance in the
compensation, whether due to endogenous or exogenous causes, can result in DNA, lipids and
protein damage(6). The aim of this project is to determine whether KDM4 is sensitive to hydrogen
peroxide, and whether the epigenetic consequences of increased amounts of ROS pass through
inhibition of these histone-demethylases. Exposure to hydrogen peroxide (H2O2) and tertbutylhydroperoxide (tBuOOH) of KDM4 are to be performed in vitro on isolated KDM enzyme, and in
cells on overexpressing KDMs.
Departures from original proposal:
Due to initial variability in the assay data obtained (difficulties in obtaining consistent results
initially), and the expansion of studies on isolated KDM enzyme, the cellular part of the project was
not possible to accomplish within the 10 week project. Instead, in vitro experiments were not limited
to the determination of IC50, but extended to cleavage assays of KDM4 by H2O2.
Description of work and result assessment
(1) In vitro IC50 determination of H2O2 for isolated KDM4A. Recombinant KDM4A was exposed to
different concentrations of H2O2 and its enzymatic activity was determined using MALDI-TOF mass
spectrometry (MS) where the reaction was monitored by the removal of methylation on histones
(Figure 1). Assays were carried out at optimal cofactor concentrations, based on previous work
performed by the group, and H2O2 was added only before the enzyme, so as it would not react with
Mnica Esteban
Summer Studentship Report
the cofactors (Fe(II), 2-oxoglutarate (2OG)) beforehand. All materials used in this study were
provided by the laboratory.
-14Da
m/z
Figure 1: MALDI-TOF MS spectrum showing

demethylation of 15mer H3K9me3 (mass
1603.53 Da) by KDM4A to produce
H3K9me2 peptide product (mass 1589.51
Da). The percentage of dimethylated
peptide relative to the substrate peptide
was calculated to find the extent of
demethylation.
Figure 2. Sensitivity of recombinant KDM4A

to H2O2. The inhibition assays were carried
out on 96 well plates, 40L per reaction.
Each reaction contained 100M ascorbate,
100M 2OG, 10M Fe(II), 100M H3115K9me3 peptide, and 1 M KDM4A with
different concentrations of H2O2. All
reactions were run in triplicates at 37C and
quenched with 0.1% Formic Acid. H2O2
(17.5M) was diluted in water to give the
desired concentrations.
The designed assay produced IC50 values (M) that were reproducible (IC50 = 23.2 M and 18.23M
for reactions stopped after 5 min and 40 min (at linear range of the reaction) respectively) (Figure 2),
demonstrating that the KDM4A is highly sensitive to H2O2.
(2) Mechanism of KDM4A inactivation by H2O2
To determine the exact mechanisms of the inhibition, further assays were undertaken with variation
in ascorbate and iron concentrations to study the direct effect of peroxide on the enzyme using SDSPAGE .
KDM4A protein was mixed with components in various combinations of KDM4A, Fe(II) and ascrobate
concentrations in 10 L final volume and incubated for 90 min at 37C. Reactions were quenched
using an equal volume of 2 x sodium dodecyl sulfate (SDS) loading buffer (50 mM Tris-HCl pH 6.8,
SDS (2% w/v), glycerol (12 %), -mercaptoethanol (5%), Bromophenol blue (0.2% w/v)), then
analysed via SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Figure 3).
Resulting gels were imaged and revealed that surprisingly, H2O2 did not seem to induce structural
changes on KDM4A, whose original weight is 45kDa, as demonstrated by the lack of KDM4A
fragmentation. However, intriguingly, its incubation with highly concentrated Fe (II) and Ascorbate
(with or without H2O2) led to increased fragmentation and aggregation, which is translated by lighter
bands of 30 kDa and heavier bands of 130 kDa respectively.
Mnica Esteban
kDa
100
70
55
40
36
25
15
KDM4A
45kDa
Lane
[H2O2]
[Fe(II)]
[Asc]
2
-
3
0.02
-
4 5
0.2 - 0.2
6
2
7 8 9
20 20
- 0.2 0.2*
2 2
10 11
20* 0.2 0.02
2 2
12 13
20 0.02 0.2
2 0.2
14
20
0.2
0.2
Figure 3. Cleavage assay of KDM4 in presence of ascorbate. 15 M protein was loaded.

Concentrations listed in mM unit. * denotes assays where the reagent was added first.
The gel shows degradation and aggregation bands (lighter and heavier than 45 kDa respectively),
that increase in intensity between lanes 8 to 12, corresponding to the lanes where highly
concentrated ascorbate was added.
Future directions of the project
This project was framed within the larger project of studying the interactions of H2O2 and the 2OGoxygenases family. Studies so far have revealed different sensitivities against H2O2 for different
enzymes. The assays carried out in this project showed that KDM4A is sensitive to HO, following
the lines drawn by the HIF-hydroxylases(7). However, the inhibition assays revealed IC50 values
between 18.23 and 23.20 M, with KDM4A proving to be very sensitive to HO. This low value is
very different to the IC50 for PHD2 of 782 M (unpublished data, H.R. William), showing the potential
for differential sensitivity of 2OG oxygenases. A low tolerance of KDMs towards ROS could have
important consequences at an epigenetic level, inducing diseases or accelerating cellular apoptosis.
The cleavage assays had the initial objective of studying the possible degradation of KDM4A by HO.
However, they did not reveal any structural damage induced by ROS, its inhibitory mechanism
remains to be studied. It did show that ascorbate, which is used in the assays to maintain Fe(II),
might be a double-edged sword, as it induced structural degradation and aggregation of the protein.
In the future, it would be interesting to continue studying the mechanisms by which HO inhibits
KDM4, expanding the study to other KDMs and eventually to other members of other 2OG
oxygenase family. Furthermore, the role of ascorbate and whether it truly acts as a protector or if it
can also be damaging is also of interest for further study.
From the student: This summer studentship has provided me with the opportunity of truly
discovering a laboratory environment. Coming from a medical background, this experience has given
me the chance of working on a wide array of skills that I consider very important for my future
professional life. Having my own project assigned and feeling responsible for it has increased my
autonomy and my curiosity for the laboratory world. The atmosphere in the lab was very enjoyable,
and everyone was very welcoming and patient. The projects being followed in the group have
triggered in me a new found interest for epigenetics that I will like to follow in my future career. I
Mnica Esteban
would like to thank the Biochemical Society for this incredible opportunity, as well as everyone in
the lab for making my summer such an interesting and lively one. I would like to thank Dr Akane
Kawamura and Rebecca Hancock for their patience and countless help.
From the supervisor: Monica has demonstrated a very strong work ethic, dedication to the project
and the ability to apply critical thinking. Even though it was her first time working in a laboratory,
she was highly competent and picked up techniques very quickly. The activity assay she was using
showed variability due to the inherent instability of the protein, but she persisted and worked
through the problems to successfully obtain robust dataset for this report. During the 8 weeks,
Monica learnt a number of techniques, including enzyme assays, mass spectrometry, peptide
purification and SDS-PAGE. She produced two well-written reports (above, and a more detailed
report for the lab), and presented at the lab meeting (to approx 15 people) at the end of her project.
She was confident and delivered a professional presentation, demonstrating her clear understanding
of the subject area, her project, and discussed the significance of her results. She answered the
questions well which led to a fruitful discussion about future work / directions for this project. She
has made important contributions towards this new project and achieved a great deal in a short time
period. It was an absolute delight to have worked with Monica and she is more than welcome to
come back in the future. Im sure she will go on to achieve many things in her future studies and
career.
References
1.
Mikhed Y, Daiber A, Steven S. Mitochondrial Oxidative Stress, Mitochondrial DNA Damage

and Their Role in Age-Related Vascular Dysfunction. Int J Mol Sci [Internet]. 2015 Jan [cited
2015 Jul 31];16(7):1591853.
2.
Klose RJ, Kallin EM, Zhang Y. JmjC-domain-containing proteins and histone demethylation.
Nat Rev Genet [Internet]. 2006 Sep [cited 2014 Dec 8];7(9):71527.
3.
Hancock RL, Dunne K, Walport LJ, Flashman E, Kawamura A. Epigenetic regulation by histone
demethylases in hypoxia. Epigenomics [Internet]. Future Medicine Ltd London, UK; 2015 Apr
2 [cited 2015 Apr 7];121.
4.
Klose RJ, Zhang Y. Regulation of histone methylation by demethylimination and

demethylation. Nat Rev Mol Cell Biol [Internet]. Nature Publishing Group; 2007 Apr [cited
2015 Jun 16];8(4):30718.
5.
Kowluru RA, Mishra M. Oxidative Stress, Mitochondrial Damage and Diabetic Retinopathy.
Biochim Biophys Acta [Internet]. 2015 Aug 3 [cited 2015 Aug 10]
6.
Rhee SG, Chang T-S, Jeong W, Kang D. Methods for detection and measurement of hydrogen
peroxide inside and outside of cells. Mol Cells [Internet]. 2010 Jun [cited 2015 Jul
10];29(6):53949.
7.
Masson N, Singleton RS, Sekirnik R, Trudgian DC, Ambrose LJ, Miranda MX, et al. The FIH
hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity. EMBO
Rep [Internet]. 2012 Mar [cited 2015 Jul 6];13(3):2517.
Mnica Esteban
Targeting the NAD+ Synthetase from Mycobacterium Tuberculosis using fragment-based approaches
Student:Nikhil Sathyan*
Supervisor:Prof.Sir.Tom Blundella
Day-to-Day Supervisors:Dr.Michal Blaszczyka and Dr.Vitor Mendesa
* Dept of Life sciences,UM-DAE Centre for basic sciences,Mumbai-400098,India
a Dept of Biochemistry,University of Cambridge,Cambridge-CB2 1GA,United Kingdom
BACKGROUND AND AIMS

Fragment-based drug discovery (FBDD) has emerged as a successful method to design inhibitors for biomacromolecules of therapeutic interest. Herein, we
describe the fragment-based approach of targeting the NAD+ synthetase from Mycobacterium, an enzyme necessary for both the de-novo pathway and
salvage pathway of NAD+ biosynthesis(Fig.1.). It makes use of a screening pipeline of biophysical and biochemical techniques .A NadE-C176A expression
construct with an inactive glutaminase domain and an active NAD+ synthetase domain, was used for fragment screening. The top fragment hits from thermal
shift assay were validated biophysically by ligand based NMR techniques and by a biochemical assay.
.Temperature was lowered to 16 C and incubated overnight. Post

expression overnight , the cells were harvested by centrifugation. Cell
pellets were re-suspended in lysis buffer supplemented with EDTA-free
complete protease inhibitor cocktail (Roche). The cells were lysed by
sonication. Debris was removed by and the supernatant was passed
through a 5 ml HiTrap IMAC Fast Flow column charged with Ni. After
washing with 50 ml of HisTrap A buffer, tagged NADE was eluted with a
0-100% gradient of HisTrap B buffer. The eluate was incubated with Ulp1
protease in a dialysis bag and dialysed overnight with gel-filtration buffer
(20 mM TrisHCl pH 8.0, 100 mM NaCl, 15% glycerol). NADE was
separated from the cleaved SUMO tag by size-exclusion chromatography
(Superdex 200) and concentrated(4500 g at 4 C) using 10 kDa Amicon
Ultra concentrators.
Fig.1. De-novo and salvage pathway of NAD+ biosynthesis.

Source: NAD+ auxotrophy is bacteriocidal for the tubercle
bacilli.Catherine Vilcheze.2010.Molecular biology
2. Experimental
2.1. Cloning, test trials, Protein Expression and Purification
Prior to the start of the project, the NadE gene was amplified by PCR dire
ctly from M. tuberculosis genomic DNA and cloned between BamHI and
HinDIII restriction sites into an in-house modified pET28 vector
containing an Nterminal SUMO tag with an internal hexahistidine tag.
The C176 mutation was introduced by site directed mutagenesis.
Testing for expression of NadE was carried out in four different strains
:BL21,BL21 Star, Origami and Tuner under different conditions of
expression temperature and IPTG concentration using an in-house
protocol (M.Hyvonen et al, 2013, http://camelot.bioc.cam.ac.uk/~marko/
methods/expression_test_2013.pdf). The transformed cells were flash
frozen in LB with 50% glycerol using liquid nitrogen and stored at -80 C
for subsequent purification.
For protein expression, LB media was inoculated with scrapings from the
80 C stock and incubated overnight (37 C). This culture was then used to
inoculate 2xTY media for expansion at 37 C until cultures reached an OD
600 of 0.4-0.5. These were then induced with IPTG (400 mM)
2.2.Thermal shift assay:Fluorescence based thermal shift assays were

performed using Bio-Rad CFX Connect real-time PCR machine to
identify hits. Each 25 l reaction mixture in 96-well plate format
contained 5 M NADE, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl,
2.5Sypro Orange dye, 5 mM test fragment and 5% DMSO (fragment
were prepared in DMSO). The temperature of the sample was raised in
0.5C increments from 25C to 90C and the fluorescence measured at
each step, with excitation/emission wavelengths of 490/575 nm.
2.3.Ligand based NMR(STD and CPMG) :Ligand based 1H-NMR

experiments were performed at 278 K on a Bruker AvanceIII AV600
equipped with a 5 mm QCI cryo probe. Samples (550 l) containing 3
mM fragment and 1% (v/v) DMSO-d6 in the presence and absence of 10
M NADE was prepared in NMR buffer (50 mM Tris-HCl (pH 8.0), 50
mM NaCl) and loaded into 3 mm NMR tubes.. The resulting spectra were
analysed with Bruker TopSpin software.
2.3 .NADE Biochemical assay

A biochemical assay of the synthetase domain of NadE was optimized on
a BMG Pherastar plate reader based on the previously published work of
LaRondeLeBlanc et al. (3). The coupled assay consisted of two steps
: the synthetase reaction where NaAD is converted to NAD+ by NadE
and the dehydrogenase reaction where alcohol dehydrogenase converts
produced NAD+into NADH, the presence of which can be detected by
measuring absorbance of the reaction mixture at 340 nm. Each 100ul of
the inhibition reaction consisted of 1mM ATP, 4mM Mg2+, 1mM NaAD
, 1mM DTT and 2.67 uM of NadE. For IC50 calculations, the inhibition
of NadE due to binding of the ligand was measured at various
concentrations of the compounds.
3. Results and Discussion

The expression trials showed that at 37 C a large amount of the
recombinant NadE form inclusion bodies compared to 17 C samples.
Based on the visual analysis of fractions on a 12 % SDS- PAGE gel
(Fig.3.), Tuner(DE3) is the best system for obtaining pure NAD+
synthetase at a growth temperature of 17 C and IPTG 400uM.
Expression of N-terminal SUMO-tagged NadE-C176A in Tuner (DE3)
gives a higher yield (6 mg of protein per litre of cell culture) than that
originally reported by Bellinzoni et al. who used a thioredoxin-NadE
fusion protein in E. coli Origami(DE3) and reported a yield of 4 mg of
protein per litre of cell culture. This is may be due to the fact that Tuner
cells produces a concentration-dependent, homogeneous level of
induction and thus helps in increasing protein solubility.
Bl21(DE3)
TUNER
BL21(STAR)
ORIGAMI(DE3)
NadE-SUMO
(90 KDa)
Fig..4.No of hits corresponding to the thermal shift observed.

Thermal Shift values shown are absolute values. All the hits caused
a negative thermal shift indicating a destabilization of the enzyme.
M
1
400
2
800
3
400
4
800
5
400 800
6
7
400
8
800
9
Fig.3. Expression test trials of NadE at 17 C . Soluble fractions

were visualized on Coomassie stained 12% SDSPAGE;
recombinant enzyme is indicated by the arrow. Lanes: 1, molecular
weight markers (Bio-Rad); 400 and 800 indicates the amount of
IPTG(uM)
A thermal-shift screen was carried out using a library of 1152 fragment

molecules. After analyzing the Tm data any fragment displaying
thermal shift (Tm) of more than 3 C was classied as a hit. In total, 47
fragment hits were identied. All the hits identified from the screen
showed negative thermal shifts (Fig..4.).Out of these, 12 fragments
showed a negative shift of value more than 6 C. These large negative
thermal shifts probably have resulted from the octameric nature of
NadE, and binding of these ligands may have disrupted/destabilized the
quaternary structure. The top six hits were screened by thermal shift
assay with varying concentration of ligands and increase in the shift in
melting temperature with respect to ligand concentration was
confirmed for all(Fig.5.).
Fig.5..Thermal shift experiments with varying concentration of

ligands.
Each
data point is an average of three independent
experiments in which every condition was reproduced thrice. Error
bars shows the standard deviation.
Ligand
288
352
527
658
744
IC50 (uM)
1000
675
2168
980
822
Tm (C)
-7
-18
-7.5
-7
-11.25
Table.1. IC50 values of ligands determined by biochemical assay.

Thermal shift values are given for ligand concentration at 5mM
and NadE at 5uM.
NMR 352 seems to be allosterically inhibiting NadE since the
concentration of the substrate (1mM) is considerably high compared to
the IC50 value (675uM). This needs to be verified through kinetic
experiments.
Fig.6.Ligand based NMR for fragments. (A) STD for NMR 352
bound to NadE. (B) CPMG for NMR 352(red) and NMR 352
bound to NadE (blue line).The flat blue line for CPMG indicates
that the signal obtained when NMR 352 is bound to NadE is
negligible in comparison to NMR 352 alone in the solution.
The top six hits were further validated by ligand based NMR
techniques namely STD (saturation transfer difference) and
CPMG(Carr-Purcell-Meiboom-Gill). In STD, if the fragment binds to
the target protein, the buildup of NOE that is transferred to the ligand
results in enhanced signal corresponding to the resonances of that
ligand in the STD spectrum. Here a representative STD NMR spectra
for ligand no.352 is shown (Fig.6.A). In CPMG, when a fragment binds
to the protein, resonances due to the fragment protons broaden and a
decrease in signal is expected. For ligand no.352 it can be seen that the
signal is almost a flat line indicating a very strong binding(Fig..6.B)
The biochemical assay also agreed with this result as NMR 352 gave
the strongest inhibition (Table.1).
Summary and Future directions

In summary, this project has helped in discovering inhibitors of Mtb
NadE. Also the optimised biochemical assay can be used for screening
the library for any ligands causing positive thermal shifts which may
not have been detected with thermal shift assay. This would both detect
hit fragments as well as allowing fragments to be ranked according to
inhibitor potency.
However the affinity of the fragments and the how and where it binds
needs to be determined by techniques like ITC and X-ray
crystallography as both these factors are very important for enhancing
the potency of the hit fragment .
Value of the studentship to the student:
Before this summer studentship I didnt have the confidence to enter
into a PhD program, but after the research experience , exposure and
the brain storming sessions with my team in Prof.Tom Blundells lab, I
cant wait to get into a research program of my choice .This studentship
also enabled me to make full use of the intellectual community at
Cambridge by participating in various seminars and lectures organised
in different institutes and departments of Cambridge university. All this
was possible with the generous support from the Biochemical Society. I
hope many other organisations follow the example of the Biochemical
society and open their doors to students of all nationalities.
-Nikhil Sathyan
Value of the studentship to the lab:
Nikhil joined the research team funded by the Gates Foundation to
work on Fragment-Based Drug Discovery targeting Mycobacterium
tuberculosis. We selected the synthesis of NaD as an important enzyme
pathway, focusing on the enzyme NaD synthetase. He developed an
expression construct of NaDE-C176A to provide an inactive
glutaminase domain and an active NaD synthetase domain. He then
developed fragment-based screening using several biophysical assays
including thermal shift and ligand-based NMR. Most significantly he
developed a biochemical assay for the system.
Fig.7. Inhibition of catalytic activity of NadE by fragments:

representative examples of dose-response curves for NMR 352 and
NMR 658.
Nikhil is a very thoughtful and bright scientist, much more relaxed and
confident than many other Indian visitors much senior to him who have
joined our research team. He very quickly became a full member of the
group focusing on the multi-disciplinary approach in fragment based
drug discovery, learning techniques quickly and working closely with
his day-to-day supervisor Dr. Michal Blaszczyk. Michal and I feel that
his contributions were significant and he will be included on a paper
that we eventually publish on this target. He has been of real value to
the progress of the research team.
Thermal shift and Ligand based NMR characterize the protein ligand
interactions on the basis of their physical binding and does not give an
exact idea about the inhibitor potency of the ligand on the enzymatic
activity of the enzyme. Hence the fragment hits was validated by a
biochemical assay. Dose-response curves were produced to determine
half maximal inhibitory concentrations (IC50s) of the top 5 fragment
hits.(fig.7,Table.1)
I had long conversations not only about the biochemistry but also about
aspects of Indian life and indeed about philosophy. Nikhil is very
independent in his thinking and has some very carefully thought-out
ideas about his research career and about life in general. This was one
of the best summer internships we have had and we were delighted that
the Biochemical Society could provide support to make his visit
possible.
-Prof.Sir.Tom.Blundell
Oliver Prosser- Biochemical Society Studentship Report
The content of my summer project departed significantly from the original proposal. The original aim
of the project was to purify and crystallise Ebola virus nucleoprotein (EBV NP), in order to gain
structural data on EBV NP using X-ray crystallography. However, prior to beginning work on the
project the structure of EBV NP core domain was solved by another group (Roa et al. 2015). Wanting
to perform a project using similar techniques, we switched the focus of the project to solving the
crystal structure of another haemorrhagic fever virus nucleoprotein, namely Lujo virus (LUJV), an
arenavirus found on the African continent.
The laboratory work associated with this project involved the expression and purification of the LUJV
NP, using Rosetta 2 cells that I transformed with a Pet 28C vector containing a sumo-His tagged LUJV
NP. Within the early weeks of the project I managed to express LUJV NP using auto induction and
purified a small amount of the protein by using affinity chromatography using Ni-NTA resin. This
involved lysing the bacteria by freeze thawing and sonication, binding the lysate to a small amount
of nickel resin in a falcon tube and then washing with of a succession of buffers containing an
increasing concentration of imidazole, preforming all wash steps using centrifugation. The protein
was then incubated overnight with sumoprotease to remove the sumo-His tag.
LUJV NP appears to be very temperature sensitive, making purification of the protein more difficult
as all buffers used needed to be at 4C during the purification. Because the buffers contained Tris,
which varies pH with respect to temperature, all buffers also had to be at the operating pH of 7.4
when at 4C.
I was able to increase the amount of protein being produced by making a glycerol stock of my
bacteria and using this to inoculate the cultures, in addition to increasing the volume of my preps
from an initial 1 litre volume to an eventual 6 litres of culture. Due the increase in volume I moved
from using a centrifuge based purification method to performing the purification using a glass
column.
X-ray crystallography requires very pure protein, so I performed size exclusion chromatography to
remove the sumoprotease and any other contaminants that remained after the nickel resin
purification. This also required concentration of the protein prep down to a volume of 5 ml. I first
attempted this using a concentrator column and centrifugation, but found that this resulted in the
loss of a large amount of protein. For later preps I concentrated using polyethylene glycol, which
proved much more successful.
As described above, the use of size exclusion chromatography resulted in the loss of a significant
amount of LUJV NP from the protein preparation. I then attempted both cationic and anionic
exchange as alternative methods for removing contaminants, both however were unsuccessful.
Another problem identified at this stage was that the Rosetta 2 cells appeared to be expressing LUJV
NP poorly. After performing a western blot I was surprised to discover that the contaminants
present in my elutions contained epitopes present in LUJV NP, suggesting that the bacteria were
producing truncated forms of the protein in addition to the full-length version. It also appeared that
small amount of LUJV NP still retained the sumo-His tag after incubation with sumoprotease,
perhaps suggesting that a small amount of the protein was misfolding.
I transformed a new set of Rosetta 2 cells and performed another western blot to check expression.
Whilst none of the new cells were producing the truncated variants of LUJV NP, expression of the
protein varied dramatically from colony to colony, although all colonies were expressing the protein
to some extent. I then used a culture that appeared to express well on the western blot and created
a new glycerol stock. Despite using this new glycerol stock and increasing the volume of culture to 10
litres I was still unable to produce enough clean protein to carry out crystallography, as a large
amount of the protein I produced was being lost during size exclusion chromatography.
For future work I would suggest increasing the amount of protein produced by the bacteria in order
to still have enough left for crystallography after size exclusion. This could involve using a different
form of induction such as IPTG. Also, growing up an overnight starter culture beforehand to
inoculate the cultures may have an impact on expression. Increasing the volume of culture appeared
to have little effect on the amount of protein purified and similarly increasing the amount of nickel
resin did not appear to increase yield.
Although I didnt manage to obtain any crystallography data for LUJV NP, my studentship has
benefited the lab by helping to optimise LUJV NP purification. For example, we now know that
polyethylene glycol can be used to successfully concentrate the protein preparation. I have also
found that individual colonies express LUJV NP at very different levels despite being transformed
with identical plasmids and being grown on the same plate. My work has also identified that
sumoprotease cleavage of the sumo-His tag is partially incomplete, likely due to mis-folding of the
protein, and that the proportion of LUJV NP that mis-folds varies between preps.
Being awarded this grant and undertaking this project has enabled me to gain experience of working
in a world class research laboratory, allowing me the opportunity to improving my practical skills,
communication in a group environment, abilities in troubleshooting problems, connections with
professionals within the field and giving me an improved sense of what it takes to have a career in
research science. I now feel I am better prepared to move to the next step when I begin a masters
degree this September and will carry the skills learnt during this studentship hopefully to a PhD and
a career in academic research.
Student: Ryan Squire

Characterisation of FeFe-hydrogenase maturation enzyme HydE
Supervisor Professor Peter Roach, University of Southampton
Background:
FeFe Hydrogenases undergo a catalytic reaction for the reversible formation of Hydrogen, where the widespread
applications of Hydrogen production from a renewable source range from transportation, coolants or use in the
chemical industry as a reagent.
The Maturation of the FeFe Hydrogenase involves the formation of the cluster HydA [1], which is thought to be
assembled by three corresponding enzymes: HydE, HydF and HydG. Much work has already been done on HydF and
HydG, with some links to their roles in the cluster formation, with HydF acting as a scaffold for the cluster to assemble
and HydG is involved in CO and CN bound ligands to the cluster. However little work has been done on the role of
HydE, where it is hypothesised that it is involved of the addition of the Dithiolate Bridge to the cluster.
[1] H-Cluster HydA, where [4Fe4S] is a Cubane structure with Iron and Sulphur.
My task for this 8 week project was to express the HydE protein in transformed E.coli, where we could then go forward
to isolate/ purify the protein, where we could carry out quantification, characterisation and potentially crystallographic
studies.
New techniques learnt on this placement included:
Agarose Gel electrophoresis
SDS-PAGE
Bradford assay analysis
Streptavidin column purification
Gel extraction procedure
DNA purification
Nucleic Acid Quantification
Small scale culture growth
Large scale culture growth (5L)
Plasmid Transformation
Using the Glove box
Liquid Nitrogen Flash freezing
Autoclave operation
Centrifuge operation
MSC operation
Enzymatic double digest
Ligations
Agar plate preparation
Results and placement progression

Weeks 1-2: transformation of the HydE Thit1265 and Thit1675 gene containing vectors into E.coli, with subsequent
double digest studies and DNA purification to purify the isolated DNA for later use.
o
Week 3: Small scale expression studies of HydE in 100 mL cultures, by incubating at 37 c until an OD600 of 0.6, then
inducing with 5 mL 20% w/v Arabinose and left to grow for 6 hours. Analysis of the cell pellet after cell lysis on the
SDS-PAGE, showed presence of HydE protein being expressed [2]:
[2]
[3]
Weeks 4-5: Large scale expression in a 5L culture gave a cell pellet of 14.6 g, this was flash frozen and thawed out in
the anaerobic glove box, where the following purification was performed.
The HydE being expressed has a Strep-tag on the N-terminus, which has high affinity for a Strep-Tactin binding
column. This meant that the sample could be washed through the column, with only the desired protein being bound
to the column, which was eluted after. This was found to be in high purity, with only one band being seen on the SDSPAGE [3].
Characterisation showed that the protein was in low concentration and was inactive after a couple of days (tested by a
o
o
SAM cleavage assay), this lead to a repeat expression, where the culture was induced at 27 c as opposed to 37 c,
and additional Fe, S and L-Cysteine were supplemented for the growth. However this provided only 13.7 g of cell
pellet, and too small a concentration of Protein to work with. Progress was also halted by inefficient column
regeneration, meaning some of the initial protein was eluted and not binding to the Strep-Tactin column.
Weeks 6-7: A new direction for this project was to attempt the co-expression of HydF and HydE proteins in the hope
that when expressed together, they would be less toxic and damaging to the cell growth culture. A vector with the
HydF gene was cut with Xho1/Nde1 enzymes where the vector fragment was isolated and the HydE 1265 and 1675
were also digested, with the smaller inserts being isolated. There were problems with the following ligation reaction, as
-1
insufficient concentration of the DNA fragments were observed, with concentrations between 2-10 ng.uL being
achieved. This was repeated several times with variable conditions on the digestion, different quantities and gel
extraction procedures. Unfortunately this lead to several unsuccessful ligation reactions between the vector and the
insert, where no growth was seen on plates with chloramphenicol and streptomycin (present in the bacteria strain and
the plasmid, therefore only resistant against both with a complete plasmid). It was concluded that the ligation was the
problem due to a control HydF plasmid growing, ruling out errors in the transformation or the plates antibiotics. No
further progress has been made on this area of the project.
Week 8: New competent cells were prepared with HydE plasmid, tested on small scale growth with induction with
arabinose at OD600, for 4 hours, found to produce HydE.
This lead to a 5L expression being performed. This unfortunately was unsuccessful due to growth beginning to occur,
then no further growth being observed.
Future directions for the project:

Isolating a larger quantity of the HydE protein will be attempted either by the co-expression with HydF, or by
optimising the growth conditions or the bacteria strain to achieve a higher yield of protein compared to what was
achieved before. Then following successful purification at a reasonable yield, to attempt various assays to find out the
mass of the protein, cleavage assays to measure its activity and potentially to crystallise the protein, with the hope that
the thiol containing substrate will be located in the active site.
Student statement:
This placement has been valuable in the sense that it has opened up my knowledge to the field of Biochemistry,
exposing me to techniques and theory that I havent experienced before. I now feel more confident in my ability to
work in a professional laboratory and have grasped a deeper concept of what it would be like to undertake a PhD,
which this has inspired me to pursue.
The project has also shown me the true value of what it is to do research, where a lot of the time things can, and will
go wrong, where you will have to try another approach and think logically to move forward, all of which makes it feel
more deserved when the results finally go your way.
I would like to thank Professor Peter Roach for allowing me into his lab and to use his resources and time, I would like
to thank both Pedro Dinis and Beata Wieckowski for spending a lot of their time in the lab teaching and guiding me
through this placement. Finally I would like to thank the Biochemical society for giving me funding to do this
placement.
Biochemical Society: Studentship report

Circadian rhythms possibly evolved through photo sensing, with the intention of minimizing UV-light
induced damage during DNA transcription. Alternative splicing (AS) is a powerful mechanism capable
of applying the information within one genetic sequence into the expression of thousands of different
proteins. These two powerful mechanisms are found universally in higher eukaryotes, emphasizing
their importance through evolution and contrarily also in instantaneous response to external stimuli.
Circadian genes function using the identical principal of negative feedback loops in clock gene
expression, driving oscillations in the levels of clock proteins with a period of approximately 24 hours.
This lab analyses the additional mechanism to expressional control of AS. The mechanism of AS
allows plants to control slightly deviant functions of AS circadian clock protein and thereby respond to
temperature shifts. (James et al., 2012) Here, we focus specifically on the light induced AS of A.
thaliana splicing factor Polypyrimidine Tract Binding protein-1 (PTB1) and briefly on LHY: a
transcriptional factor of the circadian clock.
Acquired methods and work contributed during placement

The placement consisted of assisting many aspects of the laboratory process leading up to qPCR
quantification of the plant tissue. I have successfully attempted plant growth in hydrophonics. Next,
many transgenic 3 lines (=transgenic F2) needed selection for fitness, hence the quicker sand method,
as described in Davis et al., 2009, was used. The laborious process of seed sterilization was thus not
needed, considering the likelihood of CO2 intake by micro-organisms facilitating their growth is
depressed in a sand medium. Subsequently, T3 seed growth fitness selection was carried out using
antibiotic hygromycin and analysis of successful germination. Additionally, the routine techniques of
seed sterilization and seed harvesting were also taught to me. Finally, I thoroughly learned the steps
from mRNA extraction to cDNA synthesis for quantification and the technique of qPCR.
Results
The work contributed to cDNA synthesis and qPCR were mostly related to the analysis of lightsensitive AS of splicing factor polypyrimidine tract binding protein-1 (PTB1) and LHY: a transcriptional
factor of the circadian clock. Figure 1a-b indicates there is light mediated AS of the PTB1 splice
variants SPI and SPII. There is a difference of statistical significance between the two splice variants.
SPI expression increases compared to the total PTB1 expression with decrease in light intensity from
150 to 75uE. Conversely, SPI expression decreases compared to total PTB1 when light intensity is
increased 150 to 300 uE. Hence, the reverse can be said for SPII. See Figure 1c. Figure 2 indicates
there is no statistical proof to light mediated AS of the LHY splice variants 5UTR FS and LHY I1R.
1A
1B
1C
Figure 1: a. PTB1 SPI expression increase after decrease in radiation from 150 to 75uE. SPI expression
decreases upon increase of light intensity from 150 to 300uE. b. PTB1 SPII mRNA expression increase when
light intensity is increased from 150 to 300 uE and has weak trend when light intensity decreases form 150 to
75uE. c. Splice ratio: SPI/(SPI+SPII). The total amount of PTB1 SPI increase slightly, in comparison to total PTB1,
when light intensity decreases from 150 to 75uE. The total amount of PTB1 SPI decreases, in comparison to total
PTB1, when light intensity is increased from 150 to 300uE. Difference of statistical significance between the 2
groups (the 150-75 and 150-300 groups (ZT78, 81 and 84h) as determined by one-way ANOVA: F(5,12) = 5.079,
p=0.0099.
Figure 2: There was no statistically significant difference between the 2 groups (the 150-75 and 150-300 groups
(ZT78, 81 and 84h) as determined by one-way ANOVA: F(5,12) = 0.8759, p=0.5255
Discussion and future directions

PTB1 is a splicing factor which influences AS, by association of the polypyrimidine tract to a
pyrimidine-rich sequence within pre-mRNA. (Wachter et al., 2012) PTB exploits various mechanisms
for AS, it competing with U2 auxiliary factor 65 in binding to the pre-mRNA (Saulire et al., 2006), the
U2 auxiliary factor being a non-snRNP protein is required for the binding of U2 snRNP to the premRNA branch site. PTB1 also allows for looping of RNA regions (Spellman and Smith, 2006) and its
interference with splicing factor interactions is required for exon or intron definition (Izquierdo et al.,
2005; Sharma et al., 2005).
The results describe how alternative splicing can be regulated via a gene which is itself regulated by
alternative splicing, in this case the trigger for the splicing of the splicing factor PTB1 is a variation in
light intensity. The data also indicates there is no significant evidence LHY is alternatively spliced by a
change in light intensity. Further research which could lead from this is investigation into the splicing
factors which influence splicing of PTB1 and determining whether they too are light or temperature
mediated. This could possibly enlighten whether the regulation of eventual downstream genes are
modulated by primarily one external factor or a combinatory effect of different circadian oscillations
within the environment.
Sarah Neely Biochemical Society Summer

Studentship Report Form:
Project Aims:
The primary aim of my project was to construct Multiple Antigen-Presenting System (MAPS)
conjugates from Shigella proteins to allow us to determine their potential as a vaccine against
Shigellosis in the mouse lung infection model. This research aimed to contribute to our
understanding of how, in the future, we may answer the increasing antibiotic resistance and lack of
vaccine that is currently available against Shigella. I specifically investigated serotype 5a O-antigen
and three highly conserved proteins of Shigella, MxiH, IpaD and IpaB in making the MAPS to be
tested. To produce the full MAPS constructs there were 2 main project aims: preparation of Shigella
lipopolysaccharide O-antigen and its labelling with biotin, and production of Shigella antigens as
fusion proteins with a biotin-binding moiety, rhizavidin. Upon successfully obtaining both of these
products I then aimed to assemble MAPS conjugates by a mixture of these 2, for later purification
and quality assessment.
Work done during the studentship:

To investigate the viability of Shigella proteins as conjugates for MAPS production I first
concentrated on the design and production of the fusion protein constructs. To do so the mxiH,
ipaD129-322, ipaD149-304 and ipaB were firstly isolated and inserted in the pET21B plasmid, and
fused to the rhizavidin gene. Whereas ipaB was cloned as a full length fusion protein and coexpressed to maintain solubility, two different lengths of IpaD fragments were used to establish the
most useful for later neutralising antibody generation. To then establish the base constructs these
PCR products were then ligated into the pACYC plasmid. After which I carried out expression trials of
fusion constructs in E.coli and scale-up purifications of those that were successfully expressed and
stable. Finally to confirm their abundance and purity both SDS-PAGE and Coomassie staining were
used.
At the same time, I also worked on preparation of Shigella lipopolysaccharide O-antigen and its
labelling with biotin. To do so in the time frame of the project I planned to extract Shigella flexerni
serotype 5a, detoxifiy it via the removal of its Lipid A portion, and biotinylated this product using
amine-biotin and CDAP. To plan this part of the project, with adaptations to the time frame, and
make contact with relevant persons who had previously described use of these techniques took
significantly longer than planned. Therefore, although we managed to produce a viable plan for the
desired technique, including details such as concentrations and volumes of reagents needed, we did
not have time to carry out this portion of the procedure.
Results of my research:
One of the most notable findings of the research was the fact that within the 6 week timeframe all 4
of the planned fusion constructs were successfully produced, with their sequences and therefore
identities confirmed. Although they have not yet been fused with the Shigella lipopolysaccharide Oantigen, the fusion complexes produced do provide the first step in the application of MAPS
technology to Shigella.
Departures from the original plan:

I experienced somewhat unexpected results occurring when testing the solubility of the fusion
constructs that I had produced. Despite using different methods to test the solubility of the proteins
and repeating these protocols to optimise the procedures, I still found that none of the fusion
constructs produced appeared to be suitably soluble. Importantly this highlights a fundamental need
for further research and development of this technique before MAPS technology is a viable
approach to Shigella vaccine development.
Future direction of the research:

As discussed, the apparent insolubility of the fusion constructs must first and foremost be
investigated fully. If found to be accurate, the insoluble nature of the fusion constructs produced
highlights a need for further research, to investigate the cause of this insolubility and from this
investigate the possibility of alternative routes to produce the constructs. Additionally however, the
sequence verified constructs themselves produced and the planned optimisation of the LPS
extraction method do provide a firm foundation as to the possibility and potential of MAPS
technology being applied to Shigella in the future.
Professional and personal development during the project:

I cannot fully express how lucky I feel to have had this opportunity. Not only have I really been able
to develop my confidence around the lab in techniques both familiar and new to me, but I have
gained such an invaluable insight into scientific research. An environment of asking questions, seeing
exciting possible links in the data to be explored and coming up with novel hypothesis is one which I
now fully realised that I want my future career to be in, and am determined to strive towards.
Student: Simona Andreea Vatavu

Supervisors: Professor Sherif El-Khamisy and Dr Swagat Ray
Exploiting DNA damage/repair to improve the clinical outcome of prostate cancer
Background
Several anti-cancer therapies are based on the accumulation of DNA strand breaks that
lead to genome instability [1]. . One method by which DNA strand breaks can arise is
through the abortive activity of DNA topoisomerases that usually remove torsional
stress through generating and resealing DNA strand breaks [2] . The cleavage complex
that arises as the enzymes form a covalent phosphotyrosyl bond to the DNA can become
trapped and form protein-linked DNA breaks upon collision with RNA polymerases or
replication forks [3] . Tyrosyl-DNA phosphodiesterase-1 (TDP1) is responsible for
hydrolyzing the 3-phosphotyrosyl bond covalently linking topoisomerase I to DNA
breaks, thus aiding DNA strand repair [4] . Its depletion leads to the accumulation of
TOP1-linked DNA breaks, and cells are sensitized to camptothecin (CPT), a TOP1 poison
that stabilizes cleavage complexes and induces cytotoxicity [5, 6] . Tyrosyl-DNA
phosphodiesterase-2 (TDP2) possesses robust 5 tyrosyl DNA phosphodiesterase
activity to resolve TOP2-linked DNA breaks [7] , and its depletion sensitizes cells to
etoposide, a TOP2 poison [1, 8]. .TDP2 also possesses weak 3 tyrosyl DNA
phosphodiesterase activity that can repair TOP1-induced DNA breaks when TDP1 is
absent [1] .
In hormone-driven cancers such as prostate cancer, exposure to androgens induces the
co-recruitment of topoisomerase II beta (TOP2B) and the androgen receptor to the
promoter regions of androgen-driven genes(10). TOP2B is needed for their efficient
transcriptional activation, as it mediates DNA double strand breaks to relieve torsional
stress, though its abortive activity can lead to the generation of DNA double strand
breaks(10). TOP1 is also involved in transcription, and the prevention of R-loop formation
that can otherwise lead to DSBs(11). It has previously been shown that genetic deletion of
TDP1 and TDP2 results in human, murine and avian DT40 cells becoming hypersensitive
to transcription-associated topoisomerase-linked DNA breaks[1, 9] . As prostate cancer is
initially hormone-responsive, and as topoisomerases are important in the transcription
of androgen-induced genes[ 9] , it is predicted that depletion of TDP1 and TDP2 may
result in sensitizing hormone responsive prostate cancer cells to androgen
through the accumulation of androgen-driven topoisomerase-linked DNA breaks,
thus triggering cytotoxicity.
Aims
To investigate whether depletion of TDP1 and TDP2 increases the number of
androgen-induced cytotoxic DNA double strand breaks in hormone-responsive
prostate cancer cells.
1.
2.
3.
4.
To grow androgen-responsive LNCaP prostate cancer cells in culture

To knockdown TDP1 and TDP2 separately and together through siRNA transfection
To validate knockdown by Western Blotting and TDP activity assays
To compare the number of double strand breaks in TDP proficient and deficient LNCaP
cells before and after androgen treatment, with or without CPT/etoposide by comparing
the number of H2AX foci using immunofluorescence.
Experimental design and methods

Transient transfection with siRNA and Western blotting to validate TDP1 and TDP2
knockdown
LNCaP cells (maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS) and L-glutamine at 37C in 5% CO2) were plated into 6-well plates at a
density of 0.8x105 cells/well, with the aid of a haemocytometer. DharmaFECT siRNA
transfection (with scrambled RNA, siTDP1 and siTDP2 in the corresponding wells) was
carried out when cells were 20-40% confluent, with a final siRNA concentration of
25nM. (The transfection protocol was repeated for siTDP2 after 24 hours since the
initial single hit was not successful)
Cells were incubated for a further 48 hours, after which they were washed twice and
harvested in ice-cold PBS using a cell scraper. The cells were subsequently centrifuged
at 4,500 rpm for 5 minutes at 4C, the pellet was resuspended in 30ul SDS, and the DNA
sheared using a 21G syringe. This was followed by incubation at 95C for 5 min in a
heating block, and centrifugation at room temperature, with the supernatant retained.
The samples were loaded into a 10% resolving gel with a 4% stacking gel on top, run for
10 minutes at 120mV to allow the samples move into the resolving gel, after which they
were run at 200mV for 45 minutes. The proteins were then transferred from the gel
onto a nitrocellulose membrane at mixed MW for 7 minutes, which was then incubated
with 5% milk powder and PBST for one hour in order to block non-specific binding.
A rabbit anti-TDP1 and anti-TDP2 antibody were used at 1:1000 in milk solution for 4C
overnight incubation with the corresponding membrane. This was followed by 3 washes
with PBST buffer, and incubation of the membranes with anti-rabbit secondary antibody
conjugated with HRP at 1:4000 for one hour before visualization by the ECL technique.
While the TDP1 knockdown was validated, the siTDP2 has been unsuccessful at
knocking down TDP2 even after a double hit.
Figure 1: Western blot showing that only TDP1 knockdown has been successful
Transfection with siTDP1, DHT and CPT treatments, and H2AX immunostaining assays
LNCaP cells were grown on glass coverslips

in 24-well plates, at 0.3x105 cells/well and
with 1ml full medium at 37C. DharmaFECT
transfection with 25nM siTDP1 (and 25nM
scrambled RNA as a control) was conducted
after a 24 hour incubation when 20-40%
confluence was achieved. A GFP plasmid
was also co-trasnfected at this time at
250ng/well in order to verify the
proportion of cells that have taken up the
siRNA. The full media was replaced for
hormone starvation with 5% charcoalstripped FBS media 24 hours post
transfection. The media was again replaced
two times at 24-hour intervals, the second
time immediately prior to
dihydrotestosterone (DHT) induction at
100nM. CPT was directly added to the wells
at 10uM for one hour, after a one-hour
incubation with DHT.
Figure 3: Experimental set-up
The immunostaining protocol was carried

out at room temperature. Coverslips were
washed 3x with ice-cold PBS and cells were
fixed with 10% paraformaldehyde for 10
minutes. They were again washed 3x with
ice-cold PBS and subsequently
permeabilised with 0.25% Triton X-100 for
2 minutes, followed by another 3x PBS wash
and blocking of non-specific binding
through a one hour incubation with 3%
bovine serum albumin (BSA) in PBS.
Cells were then incubated for one hour with
mouse anti- H2AX monoclonal antibody
diluted 1:1000 in 3% BSA, washed with icecold PBS, followed by a one-hour incubation
with Alexa Fluor 555 goat anti-mouse
secondary antibody and DAPI stain at
1:1000. Cells were washed with ice-cold
PBS and the coverslips were mounted to
glass slides with Immu-Mout (Thermo
Scientific). 100 cells were scored at random
from each coverslip, to obtain the average
number of DNA double-strand breaks per
LNCaP cell by counting the number of
gamma-H2AX foci using an epi-fluorescence
microscope.
Results
Figure 4: immunofluorescence staining

of transfected LNCaP cells , DAPI (blue),
GFP (green), H2AX foci (red).
Av. No. of -H2AX foci/LNCaP cell
Quantification of Double Strand Breaks

25
20
15
10
5
0
Figure 4: Graph showing the average number of H2AX foci/LNCaP cell s.e.m. of four
biological replicates
T-tests: sample
P-value
scr vs scr, DHT

0.018766975
scr, DHT vs siTDP1, DHT
0.107800908
scr, DHT + CPT vs siTDP1, DHT + CPT
0.001250194
scr, CPT vs scr, DHT + CPT
0.112594448
siTDP1, CPT vs siTDP1, DHT + CPT
0.001930557
siTDP1 vs siTDP1, DHT
0.04102077
siTDP1, DHT vs siTDP1, DHT + CPT
0.000247387
Figure 5: statistical analyses conducted using a students t-test (two-tailed distribution,
two sample unequal variance)
Induction of TDP1-depleted cells with DHT resulted in the accumulation of significantly
more DNA double strand breaks compared to non-DHT induced TDP1-depleted cells
(P=0.04), supporting the hypothesis that DHT can sensitize androgen-responsive LNCaP
cells to androgen via the accumulation of more DNA double strand breaks when TDP1 is
depleted. Likewise, the same comparison in which both samples were additionally
subjected to the TOP1 poison CPT showed a statistically significant result (P<0.01),.
Statistically significant results were also obtained in the case of scrambled-RNA treated
cells incubated with DHT compared to scrambled-RNA treated cells with no DHT
induction, (P=0.02) and knocking down TDP1 led to a statistically significant increase in
foci compared to the scrambled control when incubated with DHT and CPT (P<0.01).
Adding CPT to TDP1 knockdown cells under DHT induction also significantly increased
the number of foci observed compared to TDP1 knockdown cells with DHT (P<0.01).
Future directions
Since the siTDP2 had not been effective in knocking down TDP2, another siTDP2 must
be ordered and tested to achieve TDP2 knockdown, after which the number of H2AX
foci must be compared in cells depleted of TDP1 and TDP2 separately and together
under DHT induction. Due to time constraints, the TDP knockdown was not further
verified by TDP activity assays, though cells were co-transfected with a GFP plasmid and
it was found that siRNA had been taken up by 78% of cells. To further reduce TDP
protein levels, genetic deletion could be carried out.
This project has provided me with training in a number of techniques including cell
culture, transfection, western blotting, and DNA break measurements using
immunofluorescence. It has also given me the opportunity to develop my skills in
planning experiments and troubleshooting, as well as other transferable skills including
project management skills, communication skills, independence and also learning to
record and present data effectively. Weekly lab meetings helped me to learn more about
each lab members research and more about the field of DNA repair as a whole, and I
was also given the opportunity to learn other techniques including Northern blotting
and immunoprecipitations. This studentship has been exceptionally valuable in
strengthening my desire to pursue a PhD and to work in scientific research.
Supervisors comments
Dr Swagat Ray (day-to-day supervisor): Simonas time in the lab has been very well utilized
both in terms of generating interesting data for her project and learning varied experimental
techniques while shadowing other senior members in the laboratory. She has shown a keen
interest in how things work in a laboratory set up, including day to day running of routine
experiments like Western Blotting, protein immune-precipitation, RT-PCR and other
biochemical techniques. Her analytical skills are showcased in how she approached each
data set and tried to explain the experimental findings in a logical fashion. I hope that this
experience would give her enough encouragement to pursue a career in research.
Sherif El-Khamisy (PI): It was a pleasure to see Simona flourishing in the lab. She has been
very committed, driven and able to swiftly grasp both the intellectual and technical aspects
of the project. Simona generated high quality data in such a short time in the lab and she
continued analyzing and writing her report and a literature review for the most part of the
summer holidays. It was a pleasure supervising Simona and I look forward to witnessing her
continued successes.
References
1. Zeng, Z. et al. (2012). TDP2 promotes repair of topoisomerase I-mediated DNA damage
in the absence of TDP1. Nucleic Acid Research. 40(17) pp. 8371-830
2. Pommier, Y., et al., (2010). DNA Topoisomerases and Their Poisoning by Anticancer and
Antibacterial Drugs. Chemistry & Biology 17, pp. 421-433
3. Nitiss, J. (2009). Targeting DNA topoisomerase II in cancer chemotherapy. Nature
Reviews Cancer 9, pp. 338-350
4. Zhou, T., et al. (2009). Tyrosyl-DNA phosphodiesterase and the repair of 3phosphoglycolate-terminated DNA double-strand breaks. DNA Repair 8, pp. 901-911
5. Alagoz, M. et al. (2013). TDP1 deficiency sensitizes human cells to base damage via
distinct topoisomerase I and PARP mechanisms with potential applications for cancer
therapy. Nucleic Acids Research. 42(5) pp. 3089-3103
6. Solier, S. et al. (2013). Transcription Poisoning by Topoisomerase I Is Controlled by Gene
Length, Splice Sites, and miR-142-3p. Cancer Research 73, pp. 4830-4839
7. Ledesma, F. C., El-Khamisy, S. F. et al. (2009). A human 5-tyrosyl DNA
phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature. 461, pp.
674-678
8. Zeng, Z. et al. (2011). TDP2/TTRAP Is the Major 5-Tyrosyl DNA Phosphodiesterase
Activity in Vertebrate Cells and Is Critical for Cellular Resistance to Topoisomerase IIinduced DNA Damage. Journal of Biological Chemistry. 286(1), pp. 403-409
9. Gomez-Herreros, F. et al. (2014). TDP2 protects transcription from abortive
topoisomerase activity and is required for normal neuronal function. Nature Genetics.
46, pp. 516-521
10. Haffner, M., et al. (2011). Transcription-induced DNA double strand breaks: both
oncogenic force and potential therapeutic target? Clinical Cancer Research. 17, pp. 38583864
11. Ashour, M., et al. (2015). Topoisomerase-mediated chromosomal break repair: an
emerging player in many games. Nature Reviews Cancer. 15, pp. 137-151
Stephen Power
Membership Number: 1068995
Investigation of the Relationship between Insulin

Resistance and Fyn during the development of
Supervisor: Dr Cora ONeill
Student: Stephen Power
Background
The effects of Alzheimers disease (AD) are well
documented but there is much to learn regarding
disease etiology. Research in laboratories,
including the UCC neurodegeneration lab, has
shown that neurons become resistant to insulin in
AD at early stages of the disease [1]. This insulin
resistance is characterised by abnormally high
levels
of
insulin
receptor
substrate-1
phosphorylated at serine 616 (IRS-1 [pS616]).
Insulin resistance occurs at very early stages of
AD, is found in neurons with the defining tangle
pathology of AD and can be caused by the build-up
of amyloid beta peptide which is believed to be the
primary initiator of AD. There is a lot to be learned
about the cell and molecular mechanisms that cause
and result from the development of neuronal
insulin resistance in AD. Preliminary data from the
ONeill lab at UCC indicates that neuronal insulin
resistance in AD may link to defective activation of
the Src kinase Fyn which has also been strongly
linked to the development and pathogenesis of AD,
but mostly in animal and cell models of AD.
Exploring the functional link between Fyn and
insulin resistance heightens the capacity to
understand early signalling causes of AD, to best
develop better AD diagnostic and therapeutic
systems
Aims and Objectives

Most of the work on Fyn in AD emanates from
preclinical AD animal models and cell culture
work. However, very little is known about Fyn
integrity in AD brain, with just two published
papers existing, nor has the relationship between
Fyn and insulin resistance been explored The aim
of this project was to use cell and molecular
approaches to investigate the relationship between
neuronal insulin resistance and Fyn in the AD brain
compared to normal aged brain.
Alzheimers disease
Student Report 2015
Description of Work
Human brain tissue was obtained in an established
collaboration with the Netherlands Brain Bank.
Temporal cortex lysates and formalin-fixed paraffinembedded sections of the hippocampus and temporal
cortex from AD (n=7) and control cases (n=7) were
used in this research.
Initially,
the
study focused
on
western
immunoblotting analysis of Fyn and IRS-1 [pS616]
levels in total homogenate, membrane (100,000 x g
pellet) and cytosolic fractions (100,000 x g
supernatant) lysates from the control and AD cases
previously prepared in the lab as described [1]. These
nitrocellulose blots were re-probed with -actin to
verify the quality of protein loading. Control and AD
membrane preparations were also analysed for total
human tau (HT7) and pathological paired helical
filament (PHF-1) tau
This research also utilised double immunofluorescence microscopy to determine the
relationship between Fyn and IRS-1 [pS616] in
control and AD hippocampal and temporal cortex
brain sections. The Leica DMI3000b was used to
observe immunofluorescence alongside the Leica
Application Suite to capture images. Adobe
Photoshop allowed the green and red channels of the
image to be layered to allow visualisation of colocalisation.
Results
Comparative Western immunoblot analysis revealed
that IRS-1 [pS616] levels increased in AD compared
to matched control cases in the homogenate and
membrane (Fig 1) fractions as previously described
in the ONeill lab [1].
Stephen Power
kDa
kDa
IRS-1
250
616
[pS ]
-Actin
250
10
0
50
25
Figure 1: Levels of IRS-1 [pS616] in membrane lysates from
Alzheimers disease (AD) n=7) and control(C) brain
(n=7) showing increased IRS-1 [pS616] in AD cases
compared with controls
Levels of Fyn appear unchanged when comparing

control and AD samples for both homogenate (not
shown) and membrane fractions (Fig. 2) This
result is interesting, as further analysis in the lab
indicated levels of cytosolic Fyn decreased in AD
cases compared with controls (Fig. 3).Together this
indicates that the majority of Fyn in AD cases
localises to membrane fractions whereas the
cytosolic component of Fyn diminishes in the
disease, which could be associated with increased
activation of Fyn in Alzheimers disease.
Fyn
59kDa
-Actin
Figure 2: Levels of Fyn in membrane lysates in

Alzheimers disease (AD) (n=7) and control (C brain
(n=7)
Figure 3: Reductions in cytosolic Fyn in AD (n = 7) compared

to control (n =7) temporal cortex brain samples
Western blots of membrane fractions probing with

PHF-1 primary antibodies revealed that PHF-1 was
entirely absent from control brain, while high
amounts of PHF-1 were visible within the AD brain
lysates (not shown). HT7 primary antibodies,
which detect total human tau, showed that 6
isoforms of human tau were visible in both the
control and AD brain (Fig. 4). However, the AD
tau was increased and showed a higher molecular
weight than the control tau due to the characteristic
hyperphosphorylation of tau as part of AD
pathogenesis. The presence of high molecular
weight tau immunoreactive banding was noted in
AD which is likely to be higher molecular weight
tau aggregates.
Figure 4: Levels of total human tau in membrane lysates

in Alzheimers disease (n=7) and control brain (n=7)
Together this immunoblot analysis showed

increased insulin resistance in AD, typified by
increased levels of IRS-1 (pS616) associated with
reduced levels of cytosolic Fyn although overall
levels of Fyn or Fyn levels in membranes did not
change in AD. This changed cytosolic/membrane
partitioning of Fyn in AD cases tentatively
indicates insulin resistance in AD may be linked
with increased Fyn activation.
Immunofluorescence microscopy revealed an
increase in the levels of IRS-1 [pS616] as previously
shown in the lab [1], but also an increase in Fyn
expression in AD neurons compared with control
neurons (Fig 5). Also evident was the strong colocalisation of Fyn and IRS-1 [pS616] in AD
neurons. This result does not quite agree with the
results of the western blot which suggested that Fyn
levels were not increased in AD but rather altered
in their membrane to cytosolic expression. This
may be explained by the use of different antibodies
for immunoblotting and immunofluorescence
where the antibody used for immunofluorescence
may detect both active and total Fyn. Future studies
are investigating this further. Together the results
reveal that alterations in Fyn in AD are typified by
increased cytosolic to membrane migration of this
Src kinase that is found concurrent with, and can
co-localise with, increased neuronal resistance.
This gives a preliminary indication that increased
activation of neuronal Fyn and insulin resistance
may be mechanistically linked in Alzheimers
disease.
Human
Tau
Stephen Power
616
IRS-1 [pS
Fyn
IRS-1 [pS
Control
Fyn
616
AD
Figure 5: Immunofluorescent microscopy of the

CA1 region of control and AD
hippocampus. Co-localisation of Fyn
and IRS-1 [pS616] within neurons is
evident
Benefit to the Laboratory
My experience in the lab has been truly invaluable.

The Biochemical Society summer studentship has
provided me with the unique opportunity to hone
and expand upon my laboratory practical skills and
my theoretical knowledge of Alzheimers disease
and its mechanisms of pathogenesis. This research
experience has allowed me to gain confidence
working in a research environment, and working
here in the BioSciences Institute has provided me
with the opportunity to attend several seminars
based in the field of biochemistry. I have really
enjoyed my hands-on experience researching AD
under the guidance of Dr Cora ONeill and my time
spent here has further cemented my ambitions to
pursue a PhD following the completion of my
degree course. I am very thankful to the
Biochemical Society for providing me with this
opportunity.
Stephen was an excellent summer student,

extremely motivated and enthusiastic and a great
addition to the lab. His work over the 8 weeks was
valuable in many ways and gave us significant
preliminary data indicating that insulin resistance in
AD neurons may be linked to changes in Fyn
activation that have been described in the disease.
Stephens data will form part of future studies in
our lab in this area. We are very grateful to the
Biochemical Society for providing the funding to
support Stephen.
Bibliography
[1] Moloney, Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in AD indicate possible
resistance to IGF-1 and insulin signalling, Neurobiology of Aging, vol. 31, pp. 224-243, 2010.
Alzheimer's Association, 2014 Alzheimer's Disease Facts and Figures, Chicago, 2015.
Can N-Srcs help re-program fibroblast cells into neurons?

Victoria Scott- Summer placement in Evans lab, University of York 2015
Aim: The conversion of fibroblasts to neurons (induced neurons, iNs) has the potential to treat neurodegenerative
diseases, and thus research in this field is very important. Recent work in the Evans lab has found that transfecting
fibroblast cells with N2 Src, a tyrosine kinase, can differentiate them to have a neuronal phenotype, becoming iNs. In
addition researchers have found that several transcription factors, including Ascl1, are important for the differentiation
of iNs, (1,2). So the aim of this project was to determine whether the transfection of Cos 7 cells with N Srcs along with
Ascl1 can increase the complexity of iN morphology. In addition, I aimed to confirm the neuronal status of N-Src-iNs
by western blotting.
Work carried out:
Cell culture and transfections
Initially I spent much of my time working out the optimal conditions for transfection efficiency and cell health. When I
had found the best conditions, I transfected Cos7 cells with N1, N2 and C Srcs, alone and with Ascl1, as I wanted to
see whether Ascl1 would increase the neuronal phenotype. I repeated this experiment so I had at least 50 cells for
each condition to analyse. I then spent a few days learning how to use the fluorescent microscope and taking pictures
of my cells I had stained with antibodies.
Western blotting
I then lysed cells transfected with the same conditions and used western blotting to analyse whether there was a
change in Src and Ascl1 protein when transfected singly and together. I also used a pY146 antibody as this sight is
auto phosphorylated when Src is active.
Cloning
Alongside this experiment I used two different methods to attempt to clone Ascl1 into a bacterial vector, but
unfortunately this was unsuccessful.
North of England Cell Biology meeting
I attended the Biochemical Society sponsored NECB meeting at the end of my placement and had my first experience
of a scientific conference, I was inspired by the research of the young scientists who presented their work.
Results:
33
44
555
74.70
Percentage of cells with neurites
2500
67.90
Mean Cell body area (M2)
Mean no. of neurites per cell
1.5
65.91
1.0
65.71
61.11
58.33
0.5
54.74
27.54
Transfection condition
2000
1500
1000
500
2 3 Transfection
7 8
4 5 6 condition
1 2
5 6
7 8
Figure 1: Transfection with Ascl1 alone and in combination with Src kinases changes the morphology of Cos7 cells.
A: Fluorescence microscopy images taken with a 40X lens. Images are representative of the total sample from two Cos7 transfections of at least
50 cells from 20 fields of view. Scale bars represent 20 microns.
B: The mean number of neurites per cell at each of the transfection conditions. Error bars represent 2X standard error of the mean.
C: The percentage of cells with neurites.
2
D: The mean area of the cell body (M ). Error bars represent 2X standard error of the mean.
Transfection conditions: 1= Ascl1 + Empty GFP. 2= Empty GFP+ Empty Flag. 3= N1 Src+ Empty GFP. 4= N1 Src+ Ascl1. 5= N2 Src+ Empty
GFP. 6= N2 Src+ Ascl1. 7= C Src+ Empty GFP. 8= C Src+ Ascl1.
Empty Flag+ Empty GFP
C+ Ascl1
N2+ Ascl1
N1+ Ascl1
C+ Empty GFP
N2 +Empty GFP
N1+ Empty GFP
Ascl1 +Empty Flag
There was a significant difference between the mean number of neurites per cell in the different transfection
2
conditions, (Kruskal wallis: =34.49, df=7, p=0.00), however pairwise comparisons showed the only significant
differences to be between all of the transfection conditions and the control, except C Src+ Ascl1. This suggests that
the addition of Src kinases and Ascl1 changes the morphology of Cos7 cells into a neuronal phenotype but they are
equally effective in doing this. It also suggests that the co-transfection of Srcs and Ascl1 is not significantly improving
the complexity of neuronal morphology compared to Ascl1 alone. There was also a significant difference between the
2
mean cell body area of cells transfected in the different conditions, (Kruskal wallis: =24.07, df=7, p=0.001), but
pairwise comparisons showed this to only be between the cells transfected with Ascl1+ Empty flag and Empty flag+
empty GFP, thus suggesting that Ascl1 alone can reduce the size of the cell body. Previous work on this project in the
Evans lab has found N Srcs to reduce cell body area, so perhaps more repeats are necessary.
Flag 60.5 kDa

Src-pY146 60 kDa
Ascl1 35 kDa
GFP 27 kDa
Actin 42 kDa
Figure 2: Cell lysate western blotting
Cells were lysed after transfection with the above plasmids for 24 hours. They were then blotted with various antibodies before being
exposed on X ray film for approximately 15 minutes. Actin acts as a loading control. Ascl1 was detected at a very low level in the
C+Ascl1 GFP lysate, but it is difficult to see on this image.
The western blots suggest that when Cos7 cells are co transfected with Ascl1 and C or N1 Src, the amount of Src
kinase produced is increased, this could explain the neuronal phenotype. However C Src protein has been detected to
be the highest concentration in the cell lysate despite the cells transfected with this kinase having the least neuronal
phenotype. pY146 is auto phosphorylated when Src is active, however I believe the results are inconclusive and such
they need to be repeated. Ascl1 was detected in all of the cell lysates when co transfected with Src, however it was
not detected when alone, this suggests an interaction between the expression of Ascl1 and Src kinases. However it is
possible that there is less protein in the lysate for Ascl1 alone, as shown by the weaker actin signal.
Departures from the original proposal and future directions for the project:
In addition to the original aim, I attempted to clone Ascl1 into a bacterial vector in order to complete a kinase assay.
However this was unsuccessful by using both restriction enzymes and PCR. This part of my project will be continued
by my Supervisor, who will hopefully be more successful! Also due to time restrictions, I did not blot for neuron specific
proteins and thus I was unable to conclude whether the cells I have analysed are Cos7 cells with neurite growth or
whether they have differentiated into neurons so I think the future direction of this project would be to distinguish
between these. In addition, it would be interesting to discover whether different Srcs co-transfected in Cos7 cells could
increase the complexity of neuronal morphology, and whether they are involved in the neuronal differentiation at
different time points.
Value of the studentship to me and Career aspirations:
I have fully enjoyed my time in the lab and I feel I have learnt not only multiple important biological techniques but I
have gained a real understanding of the day to day running of the lab and what a career in research is like. This
placement has allowed me to put my knowledge into practice and has immensely improved my practical skills and so I
am now ready to begin my final year project. I am also enthused to start a career in science so at the end of my
degree I will be applying for positions in this field. I would like to thank Gareth, Sarah, Laura and Ins for all their help
and support during my project. I am also very grateful to the Biochemical Society for this scholarship.
Value of the studentship to my supervisor:
It was a real pleasure having Victoria in the lab for her 6 week studentship. She achieved a lot in a short time using a
wide range of techniques and was pretty independent towards the end. She has been able to validate the preliminary
experiments of a previous project student, which has provided us with sufficient evidence to take the project further in
the future. Although Victoria's attempts to prepare some new bacterial expression plasmids did not come to fruition,
she has made reagents that we can use to finish the cloning. The whole experience has been highly beneficial to the
lab and to Victoria's aspirations to pursue a research career.
References
1. Vierbuchen T et al (2010) Direct conversion of fibroblasts to functional neurons by defined factors, Nature.
463(7284):1035-41
2. Wapinski OL et al (2013) Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons, Cell.
155(3):621-35
Biochemical Society Studentship Report 2015

Vitalii Mudryi
Supervisor: Dr. Shozeb Haider, University College of London School of Pharmacy
Investigating structure phenotype relationship in Apparent Mineralocorticoid Excess Disease
Introduction
Apparent Mineralocorticoid Excess (AME) is a rare autosomal recessive genetic disease caused by mutations in the
11-hydroxysteroid dehydrogenase type 2 gene (11BHSD2) leading to a deficiency in the enzyme. The 11BHSD2
enzyme belongs to the short-chain dehydrogenase/reductase family (SDR) and is a 405 amino acid protein of 42kDa
that catalyses oxidation of cortisol to inactive metabolite cortisone in minaralocorticoid target tissue, mostly in distal
nephrones. This protects the mineralocorticoid receptor (MR), which has equal affinities for both cortisol and
aldosterone, from excessive stimulation by cortisol which circulates in the body at 100-1000 times higher concentration
than aldosterone. The mechanism allows aldosterone to bind to the MR receptor with consequent downstream effects on
transcription. Activation of MR receptors leads to the expression of proteins regulating ionic and water transport,
mainly epithelium sodium channel or EnaC, Na+/K+ pump. In case of deficiency of enzyme 11BHSD2 MR receptors
are highly activated that increases renal sodium reabsorbtion and potassium extraction. Low sodium level means blood
volume expansion and increase in pressure. This resemble an increase of blood pressure caused by aldosterone excess,
this is why disease was called AME. [1-3]
Aims
Aim1.Construction of the next generation of structural model of enzyme 11BHSD2 with coenzyme NAD+ and cortisol
as ligand
Significant structural conservation is observed in the SDR family of proteins. This allowed us to construct a reliable
homology models. Firstly we have to choose our templates. We took to account features such as maximum sequence
overlap, resolution of the template structure, presence of coenzyme and ligands, possibility of a dimer formation, lack of
missing regions etc. In order to define maximum overlap we used BLASTp tool to search the PDB database. Our
template sequence was extracted from UniProt database (DHI2_HUMAN (P80365)). From the BLASTp result the top
three templates that were selected were two isoforms from 17-hydroxysteroid dehydrogenase type 2 (17BHSD2) and
11-hydroxysteroid dehydrogenase type 1 (11BHSD1). Due to the lack of structural resolution for the N- and Cterminal regions in the templates, the model had to be constructed within 81- 370 residues. The best query cover was
89% for 17-hydroxysteroid dehydrogenase type 2, with 29% identity E-value 3e-15 (pdb id 1IOL). For 11hydroxysteroid dehydrogenase type 1 the cover was 61%, identity 23% ,E-value 1e-11 (pdb id 3PDJ). Besides, 17hydroxysteroid dehydrogenase type 1 had NAD+ as coenzyme bound in the structure, which would be transferred from
the template to the model. The models was constructed using Modeller software (Mod9.14) [4]. Both monomer (active)
and dimeric (inactive) forms of the enzymes were constructed. The three templates that were chosen were PDB id
1IOL, 1JTV and 1FDV. We chose the multiple template modelling method to fill missing regions in 1JTV, while 1FDV
structure was used as the template for the dimeric form of the 17BHSD2 enzyme. Multiple alignment of 1IOL, 1JTV,
1FDV amino acid sequences was performed using ClustalX software [5] and then refined manually to avoid gaps in
secondary structure regions. After model construction, ligands were docked in the structure. Due to structural similarity
between cortisol and estradiol, we assumed that cortisol should bind at the same site as estradiol does. Thus the receptor
binding site was defined around estradiol. To prove that cortisol has the best affinity to 11BHSD2, we docked several
other ligands (cortisone, progesterone, estrogene, testosterone), and the binding energy for cortisol was the strongest at
-23,44 kcal/mol. For this task we used ICM Molsoft Pro software [6].
Aim2.Optimisation of the model via Molecular dynamics simulations
We performed Molecular Dynamics (MD) refinement of our monomer and dimer models. For MD simulations we used
AMBER software [7], employing the ff14SB force field. Parameters for the ligands were created using antechamber
tools. The system was solved with 8 water TIP3PBOX and neutralized with 5 chlorine atoms. Firstly the
minimisation was performed, than NVT equilibration for 5ns with heating to 300K, finally MD Production NPT for 100
ns.
Figure1: 3D structures of HSD11B2 in monomer and dimer forms after Molecular dynamics simulations
Figure2: a)the model of 11HSD2 before molecular dynamics refinement b)the model of 11HSD2 after molecular
dynamics refinement. (The lowest normalized residue energy is shown by blue color, the highest one by red.)
c)superimposed a - black, b red with the RMSD value of 1.739 )
Aim3.Map known mutations and predict their effect on the structure.
Searching through databases and reading papers we created a list of mutations. The mutations were mapped on created
before structures. This allowed to see the influence of each mutation on protein features such as stability, dimer
formation, ligand binding.
Figure3: Missense mutation R186C causes disruption in R186 E190 amino acids electrostatic interaction, which is
important for dimer formation. This could be the cause of AME type1, more severe type.
The value of the studentship to the student

The studentship has enabled me to experience what carrying out research in a leading institution. It has opened for me
perspectives in structural bioinformatics. It has allowed me to use most up-to-date and popular bioinformatic tools and
what's more important how to plan an in silico experiment by myself. I am in debt to Biochemical Society for providing
me this opportunity, which has strengthened my resolve to pursue becoming a scientist as a career.
The value of the studentship to the lab
The studentship allowed the Haider lab to generate valuable structural data. The models created by Vitalii were then
used to map all known mutations of AME and provide a structural rationale for the defective enzyme. This finding was
exemplary as it helps clinicians in quickly predicting the severity of the disease by assessing these mutations. This work
is a part of a study that is being submitted to the PNAS journal.
References:
1)Blood Press.2014;23(3):189-92
2)Mol. Cell. Endocrinol 2014;384:71-82
3)Physiol Genomic 2010;42(3):319-330
4)J. Mol. Biol. 1993;234: 779-815
5)Bioinformatics 2007;23: 2947-2948
6)Proteins 1997;1:215-220
7)J. Computat. Chem. 2005;26:1668-1688
Biochemical Society Summer Vacation Studentship 2015

High-throughput screening to identify novel inhibitors of
human -methylacyl-CoA racemase 1A (AMACR; P504S)
Introduction
Student: Yoana Petrova. Supervisor: Dr. Matthew Lloyd.

Medicinal Chemistry, Department of Pharmacy and Pharmacology, University
of Bath, U.K.
Aims and objectives
Pristanic acid [2R,S,6R,10R-(2,6,10,14-tetramethyl)pentadecanoic

acid] is a 2-methyl saturated fatty acid derived from phytanic acid
[3R,S,7R,11R-(3,7,11,15-tetramethyl)hexadecanoic acid] by oxidation in the body. Phytanic acid is obtained from the diet and is
particularly abundant in foods such as red meat and dairy
1
products. The -oxidation of phytanic acid produces a mixture of
2
2R- and 2S-methyl pristanic acid epimers. However, the acyl-CoA
oxidases responsible for the further metabolism of pristanic acid by
-oxidation have the absolute requirement for the 2S-methyl acyl3
CoA esters. The enzyme involved in the conversion of the 2R to the
4
2S isomer is -methylacyl-CoA racemase (AMACR). AMACR has also
the enzyme responsible for the activation of Ibuprofen in the
pathway, converting the R-Ibuprofen to the pharmacologically
5
active S-Ibuprofen. The reactions catalysed by AMACR proceed via
6
an enolate intermediate.
AMACR levels are increased (up to 9 fold) in all types of prostate
cancers and phytanic acid has been shown to have a role in
7,8
regulating levels. Studies have shown a correlation between the
consumption of products high in phytanic acid (red meat and dairy
9,10
products) and advanced prostate cancer.
It was demonstrated
that reducing AMACR levels using siRNA reduced the proliferation
7
of prostate cancer cells, making AMACR an attractive target.
There are two main problems hindering the discovery of inhibitors
against AMACR: the lack of a convenient assay to measure the
activity of the enzyme and the fact that the CoA moiety is essential
for binding of the inhibitor. Many CoA analogues were synthesised
and showed inhibition of AMACR, however these are not drug-like
molecules due being zwitterionic and having high MWs. The first
problem was resolved when a colorimetric assay was developed by
Dr. Maksims Yevglevskis (unpublished work), who is part of Dr.
Lloyds research group. The colorimetric assay uses the reaction, in
which a colourless acyl-CoA substrate is converted to a yellow
product by AMACR (Scheme 1). The formation of the yellow
product allows the activity of the enzyme to be assayed in 96-well
plates using a spectrophotometer. The second problem concerning
the lack of drug-like inhibitors for AMACR is addressed in the aims
of this study.
The aim of the project is to discover a novel class of small

molecule compounds, which inhibit the activity of AMACR and
exhibit drug-like properties. The objectives were to: 1)
Optimise the colorimetric assay conditions for high throughput
screening of compounds against AMACR; 2) Complete high
throughput screening of 7680 compounds and identify
potential inhibitors (hits); 3) Further characterise the hits
with respect to their inhibitor properties.
Materials and Methods

All materials and reagents are obtained from the Sigma-Aldrich
Chemical Co. or Fisher Scientific Ltd unless otherwise stated. The
compound libraries were obtained from MRC Technology.
AMACR expression, extraction and purification
Competent E. coli Rosetta2 (DE3) cells (Novagen) were prepared
and transformed with plasmid encoding for human His-tag AMACR
6
enzyme using the CaCl2-heat shock method. Recombinant cells
expressing the enzyme were lysed with the One Shot cell disruption
system. The AMACR enzyme was purified by metal chelate
chromatography. SDS-PAGE was performed to confirm the presence
of the enzyme in fractions. Visking tubing dialysis was used to
buffer exchange the enzyme, and the concentration of the enzyme
was determined by measuring the absorbance at 280 nm using a
11
Helios Omega spectrophotometer .
High throughput screening assay conditions
The assay was performed in 96-well half-area plates. The activity of
the enzyme was assayed by measuring the absorbance at two
wavelengths (354nm and 390nm) using a BMG LabTech FluoStar
Omega spectrophotometer. Measurements were taken every
minute for 8 minutes. The total assay volume used was 100L. The
library compounds were used at 30M in the assay, giving a final
DMSO concentration in the assay of 3% (v/v). The library
compounds were incubated with the AMACR enzyme for 10
minutes before addition of the substrate. The substrate
concentration in the assay was 18M, which is equal to the Km
-1
value (unpublished work). The enzyme was used at 0.086mg.mL in
the assay, which is within the concentration range previously used
in Dr. Lloyds group. The positive control had 3% (v/v) DMSO to
replace the library compound and the negative control had
phosphate buffer to replace the enzyme and 3% (v/v) DMSO to
replace the inhibitor.
IC50 determination
The IC50 determination was performed on inhibitors identified from
the screening under the same assay conditions, with a substrate
concentration of 40M in the final assay. The top concentration of
the selected inhibitors in the assay was 30M. A 3-fold dilution
series was used with 8 concentrations of inhibitor in total. All
dilutions of the inhibitors were performed in DMSO in order to
avoid precipitation of the inhibitors.
Scheme 1 The scheme shows the novel E1cB reaction catalysed by

AMACR. The colourless acyl-CoA substrate is converted to a yellow
2,4-dinitrophenolate product and a colourless acyl-CoA product in
one-step irreversible reaction.
|1
Drug 1
Drug 2
0.40
0.50
0.45
0.35
0.40
0.30
A345
0.35
A354
Reversibility experiments
The assay conditions were consistent with those used in the IC 50
determination. The inhibitors were incubated with the
concentrated enzyme for 10 min. The inhibitor concentration in the
inhibitor-enzyme mixture was 30M. Before the addition of the
substrate, the inhibitor-enzyme mixture was diluted with
-1
phosphate buffer to give 0.086mg.mL enzyme and 0.66M of
inhibitor in the final assay. Rates were determined as above
0.25
Drug at 30 M
Drug at 0.66 M
Positive control
Negative control
0.20
Drug at 30 M
Drug at 0.66 M
Positive control
Negative control
0.25
0.20
0.15
0.15
0.10
0.10
0
10
Time (minutes)
Drug 2
10
Drug 4
0.40
0.35
0.35
0.30
0.30
A345
0.40
0.25
Drug at 30 M
Drug at 0.66 M
Positive control
Negative control
0.20
0.25
Drug at 30 M
Drug at 0.66 M
Positive control
Negative control
0.20
0.15
0.15
IC50 determination
The results from the IC50 determination are displayed in Figure 1.
The top concentration of inhibitors used in the IC50 determination
was 30M due to the availability of limited amounts of compound.
This is approximately 3 times lower than the top concentration of
100M used previously in the IC50 determination of various CoA
analogues, known competitive inhibitors of the AMACR. The IC50
values reported for some rationally designed 2-methylacyl-CoA
analogues, including R- and S-Ibuprofenoyl-CoA, ranged between
13, 14
0.5M and 20M.
The inhibitors reported in this study have IC50
values in that range, suggesting that they have similar potency to
the known inhibitors of the enzyme.
Time (minutes)
Drug 3
A354
High throughput screening

Possible inhibitors (hits) were identified by comparison of the
absorbance trace to positive and negative controls, and were taken
forwards for IC50 determination. Approximately 70 hits were
identified during the screening, giving a hit rate of 0.9%, which is
consistent with the hit rate of <1% expected from a diverse and
12
unbiased library.
Drug 1
0.30
0.10
0.10
0
10
Time (minutes)
10
Time (minutes)
Figure 2 Reversibility experiments curves for four selected inhibitors

(drugs 1-4). The positive control is shown in red and the negative
control in yellow. The black line represents the drugs at 30M and
the green line the diluted drug at 0.66M.
between the chemical structures of the inhibitors by
computational chemistry methods will allow the identification of
a common pharmacophore and the synthesis of analogues with
improved activity. The development of a fluorescent assay is
another future goal, as it will allow the measurement of the
activity of AMACR in cell cultures and the evaluation of the
inhibitors in vivo.
Deviations from original project
Drug 3
Drug 4
The original proposal for the project aimed for developing a

convenient assay for measuring the activity of the AMACR. The
reaction shown in Scheme 1 had been characterised by Dr.
Lloyds research group at the time the project proposal was
drafted. Therefore, the determination of the kinetic parameters
of known inhibitors and the use of the assay to determine the
inhibitors potency were part of the original project proposal.
However, by the time I started the project, that part of the work
in the proposal had been completed and the project I embarked
on was a continuation of the original one.
Value of the studentship to the student

Figure 1 IC50 curves for four selected inhibitors (drugs 1-4). The IC50
values are : 16.2M (Drug 1), 15.2M (Drug 2) 9.3M (Drug 3) and
12.8M (Drug 4).
Reversibility experiments
The reversibility experiments were performed in order to define the
mechanism of inhibition (Figure 2). The experiment required
incubation of the enzyme with inhibitor at a concentration 10 times
12
the IC50. However, due to insufficient amount of compound, 30M
of inhibitor was used, approximately 2-3x IC50. It can be seen that
the progress curve for the diluted inhibitors (0.66M) is parallel to
the one for the positive control, suggesting a reversible mode of
inhibition because the activity of the enzyme is fully restored upon
diluting the inhibitor to 0.04-0.07x IC50. The reversibility
experiments also suggest that the compounds identified are
enzyme inhibitors as opposing to non-specific denaturing agents.
Future Directions
The future directions of the project will involve performance of
the IC50 determination and reversibility experiments to all of the
hits identified from the screening. Furthermore, comparisons
2|
The 8-week placement funded by the Biochemical Society

allowed me to gain a valuable experience in variety of laboratory
techniques such as: preparation of competent cells; protein
expression using recombinant DNA technology; protein
purification using His-tag; SDS-PAGE analysis. I was actively
involved in the optimisation of the assay conditions for high
throughput screening and in the process of screening. The
placement gave me the opportunity to talk to other members of
the team and to learn what the life of a researcher entails. As a
consequence of the 8 weeks in the lab, I will definitely be
considering a PhD after graduation.
References
1) Lloyd, et al., Prog. Lipid Res., 2013, 52, 220-230;
2) Ackman, et al., Lipids, 1967, 2, 357-362;
3) Battaile, et al., Lipid Metab., 1998, 1390, 333-338;
4) Schmitz, et al., Eur. J. Biohem., 1995, 231, 815-822;
5) Woodman, et al., Chem. Commun., 2011, 47, 7332-7334;
6) Darley, et al., Org. Biomol. Chem., 2009, 7, 543-552;
7) Zha, et al., Cancer Res., 2002, 62, 2220-2226;
8) Mobley, et al., Cancer Epidemiol. Biomarkers Prev., 2003, 12,
775-783;
9) Wright, et al., Prostate, 2011, 71, 498-506;
10) Wright, et al., Int. J. Cancer, 2012, 131, 1396-406;

11) Yevglevskis, et al., Chem. Commun., 2014, 50, 14164-14166;
12) Copeland, Evaluation of enzyme inhibitors in drug discovery
A guide for medicinal chemists and pharmacologists, John
Wiley&Sons, 2005;
13) Carnell, et al., J. Med. Chem., 2007, 50, 2700-2707;
14) Carnell, et al., ChemMedChem., 2013, 8, 1643-1647.
|3
Please do not adjust margins
Biochemical Society Summer Vacation Studentship Report

Supervisors: Dr. Rachel Williams & Dr. John Taylor
Student
: Ngai Tsz Wai
Characterisation of the effect of IL-24 on human gingival keratinocytes and fibroblasts in vitro
Aim:
Our recent discoveries show a synergistic effect of IL-1 and leptin on the secretion of interleukin-24 (IL-24)
in primary gingival fibroblasts. IL-24 is a cytokine of the IL-10 family with known immunomodulatory effects,
such as, promoting inflammatory responses in epithelial cells. However, there is no information about
similar effects in gingival keratinocytes and gingival fibroblasts. This project aimed to investigate the
expression of IL-24 heterodimeric receptors consisting of either subunits IL-20R1/IL -20R2 or IL22R1/IL20R2 on human gingival fibroblasts (HGF) or primary gingival keratinocytes and to find out the effects of IL24 on these cells.
Methods:
Within the first two weeks, I looked at the expression of IL-24 receptors in primary keratinocytes. First, I
observed the culturing of primary keratinocyte samples, extracted and analysed their RNA. Then, I
converted RNA into cDNA through reverse transcription and amplified DNA segments through polymerase
chain reaction. After that, I ran 3.5% Agarose Gels using those DNA segments along with molecular size
markers at 70 Volts for 2 hours to look at the expression of IL-24 receptors. I also performed a Human IL-8
ELISA Assay to assess the effects of IL-24 on IL-8 secretion in HGF using HGF from three patients.
In the following three weeks, I looked at the expression of IL-24 receptors in HGF using the same methods
mentioned previously and performed a Human CXCL-12 ELISA Assay using the same set of HGF donors in
the Human IL-8 ELISA Assay.
In the remaining three weeks, I further ran two gels using DNA segments of three other HGF donors. I also
acquired invaluable tissue culture techniques of cells feeding and splitting as well as the actual culturing of
primary keratinocytes from donors gums.
Result:
Expression of IL-24 receptors in primary keratinocytes
IL-20R2, IL-20R1 and IL-22R1 subunits that make up IL-24 receptors are expressed in primary
keratinocytes as reflected by visible bands in the gels (not shown) at 75bp for IL-20R2, 125bp for IL-20R1 a
d 150bp for IL-22R1.
Effects of IL-24 on IL-8 secretion in HGF
Fig 1. Human IL-8 ELISA Assay (HGF donors: patient A, B and C)
HGF donors secrete IL-8 at varying concentrations under different sample conditions as shown in Fig 1.
HGF cultured from patient A secretes low IL-8 concentration with a mean of 7pg/ml under both unstimulated
and IL-24 stimulated conditions whereas HGF cultured from patient B and patient C donors secrete even
lower IL-8 concentration with a mean of 0pg/ml. In contrast, HGF from all patients stimulated with IL-1
secrete high IL-8 concentrations. HGF cultured from patient A secretes the highest concentration with a
mean of 2098pg/ml, HGF cultured from patient B secretes the second highest with a mean of
1437.374pg/ml that is slightly higher than the secretion of the HGF cultured from patient C with a mean of
1294.699pg/ml. Similar results are obtained when both IL-24 and IL-1 were used to stimulate HGFs from all
patients. HGF from patient A secretes the highest concentration with a mean of 2098pg/ml, HGF cultured
from patient C secretes the second highest concentration with a mean of 1608.71pg/ml that is greater than
T22/11 2s secretion with a mean of 979.0262pg/ml.
Expression of IL-24 receptors in HGF
Visible bands in gels (not change) are seen in all three HGF donors at 75bp for IL-20R2, 125bp for IL-20R1
and 150bp for IL-22R1 confirming that IL-20R2, IL-20R1 and IL-22R1 are expressed in HGF.
Effects of IL-24 on CXCL-12 secretion in HGF
HGF donors secrete CXCL-12 at varying concentrations under different sample conditions as shown in Fig
2. HGF cultured from patient B secretes the highest with a mean of 69.295pg/ml, HGF cultured from patient
A secretes the second highest concentration with a mean of 16.3785pg/ml and HGF cultured from patient C
secretes the lowest with a mean of 2.712pg/ml. When stimulated with IL-1, CXCL-12 is also secreted yet
with a much lower concentration compared to that of IL-24 stimulated. HGF cultured from patient B secretes
the lowest with a mean of 2.712pg/ml and HGF cultured from patient A secretes the second lowest
concentration with a mean of 5.9525pg/ml. Both HGFs cultured from patient A and B secrete CXCL-12
under stimulation of both IL-24 and IL-1 with a mean of 16.575pg/ml and 38.17pg/ml respectively.
Exceptionally for HGF cultured from patient C, a much higher CXCL-12 concentration is obtained instead
with a mean of 6.25pg/ml and no CXCL-12 is secreted when stimulated under both IL-24 and IL-1.
Fig 2. Human CXCL-12 ELISA Assay (HGF donors: patient A, B and C)
Conclusion:
The RT-PCR results confirm the expression of IL-24 receptors in both primary keratinocytes and HGF.
Overall the ELISA data suggests IL-24 stimulated HGFs do not secrete IL-8 but secrete CXCL-12 whereas
stimulation with IL-1 and both IL-1 and IL-24 results in the secretion of both IL-8 and CXCL-12 with varying
concentrations. This suggests a synergistic effect of IL-1 and IL-24 on the secretion of both IL-8 and CXCL12.
Future directions:
Further research into effects of IL-24 on human periodontal epithelia can be done using other cytokine or
chemokine assays.
Departures from the original proposal:
There is no departure from the original proposal.
Value of studentship:
Throughout the studentship, I have learnt and practiced wide range of techniques from accurate and
precise pipetting, culturing, feeding and splitting cells to running ELISA assays and agarose gels. This
opportunity has also answered my doubts regarding my ability to take up a PhD after my undergraduate
studies. I would also like to express my gratitude to my supervisor, John Taylor for his support and my
postdoc, Rachel Williams for her patience and teachings throughout these eight weeks.
Role of cdc42 targets in Cancer cells interaction with endothelial cells

Student: Yunhui Zhuang
Supervisor: Professor Anne Ridley and Dr Camilla Cerutti, Kings College London, UK
Background and objectives

Metastasis is the major cause of death in cancers. During cancer metastasis, cancer cells from the
primary tumour site invade their surrounding tissues and migrate through lymphatic or blood vessels
to enter the circulations. They can then cross through the endothelium of blood vessels and migrate
into a new tumour site where they survive and proliferate. This involves cell adhesion and
interaction of cancer cells with the endothelium in the blood vessels to allow migration and
development of secondary tumours (Reymond et al., 2013).
Rho-GTPases have been extensively studied for their effects on cell adhesion and migration through
remodelling of the actin and microtubules cytoskeletons. Cdc42 is one of the Rho GTPases that has
been found to mediate transendothelial migration of cancer cells through regulating 1-integrin at
transcriptional level (Reymond et al., 2012). IQGAP1 and N-WASP are both effectors of Cdc42
(Fukata & Kaibuchi, 2001), but their effects and roles in transendothelial migration are not yet fully
understand.
Brain metastasis from prostate cancer is infrequent, but have very poor prognosis (Tremont-Lukats
et al., 2003). Here, the aim was to study the effect of IQGAP1 and N-WASP on 1-integrin level, and
how they affected prostate cancer cells interaction with brain endothelial cells.
Methods and Materials

The prostate cancer cell line DU145 was primary used to study the effect of IQGAP1, N-WASP and
1-integrin on cancer cells interaction with hCMEC/D3 brain endothelial cells. DU145 cells were
cultivated in RPMI-1640 medium (Gibco) with L-glutamine, 10% foetal bovine serum (FBS), 100U/ml
penicillin and 50g/ml streptomycin. The hCMEC/D3 brain endothelial cell line (Weksler et al., 2005)
was grown in EBM2 medium (Lonza) on flasks coated with 5% college from calf skin (Sigma-Aldrich)
DU145 cells were transfected with siRNAs targeting IQGAP1 (GE-Dharmacon), N-WASP (SigmaAldrich) and 1-integrin (Thermo Fisher Scientific) or control siRNA in 6-well plates using
oligofectamine (Invitrogen). After 72 h, cells were lysed in sample buffer and cell lysates stored at 20C.
Cell lysate were loaded and separated on a 6% SDS polyacrylamide gel, then transferred to
nitrocellulose membrane. Membranes were blocked with 5% skimmed milk powder, then incubated
overnight with mouse anti-IQGAP1 (Invitrogen), rabbit anti-N-WASP (Cell Signalling Technology)
and mouse anti-1-integrin (R&Dsystem) antibodies, as well as mouse anti-GAPDH (Merck Millipore)
antibody (house-keeping gene as a loading control) for 1h. This was followed by washing with Tris
buffered saline with Tween 20 (TBS-T) and incubation with HRP-conjugated secondary antibodies
(GE Healthcare). Blots were developed by enhanced chemiluminescence (ECL) and exposed to the
film. Results on the film are then analysed in ImageJ software (NIH), where bands are quantified and
data are calculated against GAPDH and compared with the control.
For adhesion assays, siRNA-transfected DU145 cells were detached 72h after transfection using nonenzymatic cell dissociation buffer (Sigma-Aldrich) and labelled with cell tracker green CMFDA (Life
Technologies). For live adhesion assays, images were taken after adding DU145 cells to confluent
hCMEC/D3 cells in -slide Ibidi 8 well plates for 15 min at 37C, followed by two washes with PBS.
For each condition, 10 fields of view were acquired with a confocal microscope (Zeiss LSM510) and
the number of adherent DU145 cells were counted using ImageJ software. For fixed adhesion assays,
4% paraformaldehyde was added after incubating DU145 cells with D3 cells for 15 min in 24-well
plates. Cells were then washed with PBS and stored at 4C for imaging on Nikon epifluorescence
microscope the next day. For each condition, cells were plated in duplicate wells and 2 field of views
were taken per well.
Intercalation assays were performed by adding CMFDA-labelled DU145 cells to confluent hCMEC/D3
cells in 24 well plates in a time-lapse microscope (Nikon), where plates were kept at 37C and 5%
CO2. Images were captured every 5 min for 10 h using MicroManager software. For each condition
were plated in duplicate wells and 2 field of views were taken per well. Images were analysed using
ImageJ software and DU145 cells were counted as intercalated when they were flattened and
became part of the monolayer with hCMEC/D3 cells.
Results
Transfection of DU145 cells with siRNAs successfully reduced IQGAP1, N-WASP and 1-integrin
protein expression, in comparison to the control siRNA (Fig. 1). Two different siRNAs targeting each
gene were tested. 1-integrin antibody detected two bands by western blotting, which are most
likely be the glycosylated form and unglycosylated (lower bands) form. It was consistently observed
that knockdown of IQGAP1 and N-WASP reduced the level of 1-integrin, affecting the lower band
(unglycosylated) the most.
IQGAP1-1 siRNA reduced IQGAP1 expression by approximately 60%, and IQGAP1-3 siRNA has an
effect of 50% reduction. Both N-WASP2 and N-WASP3 siRNAs have reduced N-WASP expression
level by 40%. Both 1-integrin1 and 1-integrin2 siRNAs have reduced 1-integrin level by 90%.
Figure 1. Two representative blots were shown here. On the right, DU145 cells were transfected for 72hours
and total expression of IQGAP, N-WASP and 1-integrins were analysed in western blot. DU145 cells were
transfected with single siRNA oligos, as well as double and quadruple knock down with same total
concentration of siRNA oligos. Mock: reagents without siRNA; I+I: IQGAP1-1 and IQGAP1-3 siRNAs; N+N: NWASP2 and N-WASP3 siRNAs; I+I+N+N: IQGAP1-1, IQGAP1-3, N-WASP2 and N-WASP3 siRNAs
Figure 2. Western blot results of DU145 cells transfected with two siRNAs targeting IQGAP1, N-WASP, 1
integrin or control siRNA (control) after 72 h. Mock: cells treated with transfection reagent alone; Cells:
untransfected cells. Bars indicate mean SEM relative to control siRNA, n=3.
Experiments were performed to study the effect of IQGAP1, N-WASP and 1-integrin depletion on
cancer cell interactions with brain endothelial cells. Adhesion assays were performed, with one live,
and two fixed assays with DU145 cells on confluent HMEC/D3 cells. 1-integrin significantly reduced
adhesion of DU145 cells onto D3 brain endothelial cells by approximately 50% (Fig. 3). However,
IQGAP1 and N-WASP did not alter adhesion compared to the control siRNA-transfected cells.
Figure 3. Individual results of each adhesion assay. Exp1 was done with live cells in -slide Ibidi 8 well plates,
whereas Exp2 and 3 were fixed adhesion assay on 24-well plates with two duplications per plate.
When looked at individual results for each adhesion assay (fig.4), N-WASP3 seems to have an effect
on adhesion ability of DU145 cells on D3 cells in fixed adhesion assay of Exp2 and 3. However, this
observation is not consistent with Exp1 when images were taken live.
1.5
1.0
0.5
0.0
Figure 4. Adhesion of DU145 cells to

hCMEC/D3 cells. DU145 cells were
transfected with the indicated siRNAs.
After 72 h, they were labelled with
CDFMA before imaging. Results show
mean +/- SEM relative to control cells,
n=3.
M
O
C
K
O
N
T
IQ RO
L
G
IQ AP
G -1
A 1
PN
-W 1 3
N AS
1 WA P 2
SP
I
1 NT
3
IN EG
TE R I
G N1
R
IN
2
C
EL
LS
Adhered DU145 cells (ratio)
Summary of DU145 ADHESION TO hCMEC/D3 cells
siRNA
In parallel to adhesion assays, transfected DU145 cells were used to analyse intercalation with
hCMEC/D3 cells. Cells were imaged by time-lapse microscopy. Over 6 hour period, there was no
clear difference in intercalation of DU145 cells depleted of NWASP, IQGAP-1 or 1-integrin (Fig. 5) in
the two assays.
Figure 5. Summary of duplicate results of intercalation assays of DU145 cells on hCMEC/D3 cells. In each assays,
eight conditions were duplicated in each plates and two field of views were imaged over 10 hours with 5
minutes interval.
Discussion
Western blotting analysis showed that all IQGAP1, N-WASP and 1-integrin siRNAs were able to
reduce total protein expression levels. It was interesting to observe that IQGAP1 and N-WASP
depletion reduced 1-integrin protein levels, and especially, the unglycosylated form. This may
suggest that both IQGAP1 and N-WASP are involved in internal cell trafficking and processing of 1integrins via the same or a different pathway. Since IQGAP1 is known to contribute to exocytosis in
cells (Ory & Gasman, 2011), it could affect endoplasmic reticulum (ER)-Golgi trafficking of 1integrins. N-WASP is an actin nucleating protein which may affect vesicular delivery of 1-integrin
between the ER and Golgi. However, both IQGAP1 and N-WASP are effectors of Cdc42 (Fukata &
Kaibuchi, 2001), and Cdc42 has been found to regulate 1-integrin transcription (Reymond et al.,
2012), and thus they could also act on transcription level. Further work could be done, such as qPCR,
to determine whether IQGAP and N-WASP affect 1-integrin mRNA levels.
Integrins are cell-matrix adhesion receptors that regulate cell adhesion and transduction of signalling
pathways from the extracellular matrix to the cell (Miao et. al, 2002). From the results summary of
the adhesion assays, it is apparent that 1-integrin knock down significantly reduced DU145 cell
adhesion to hCMEC/D3 cells. This indicates that DU145 cells may use integrins to adhere to
hCMEC/D3 brain endothelial cells. N-WASP seems to reduce DU145 adhesion to hCMEC/D3 cells,
which could be due to N-WASP reducing 1-integrin levels (as suggested in western blots). However,
this needs to be repeated to determine if the effect of N-WASP on adhesion is significant.
Interestingly, 1-integrin, IQGAP1 and N-WASP knock down seem to have no significant effect on
DU145 cell intercalation between hCMEC/D3 brain endothelial cells. This suggests that DU145 cells
may utilise use a mechanism independent of 1-integrin to intercalate between hCMEC/D3 brain
endothelial cells. This is in contrast to previous observations with 1-integrin-depleted PC3 cells,
which had strongly impaired intercalation between human umbilical cord endothelial cells (Reymond
et al., 2012). This may due to different properties between human umbilical cord endothelial cells
and hCMEC/D3 brain endothelial cells. Brain endothelial cells have specialised tight and adherent
junctions that forms the blood-brain barrier (BBB). This endothelial barrier can regulates its
transcellular permeability by responding to different signalling molecules, which might be 1integrin independent. On the other hand, DU145 cells may differ to PC3 cells by using different
mechanism that is 1-integrin independent to intercalate into endothelial cells. Further study can be
done to test these hypotheses by performing adhesion and intercalation assays with PC3 cells on
hCMEC/D3 cells, and DU145 cells on human umbilical cord endothelial cells.
This summer vacation research project funded by Biochemical Society has enabled me to work at
cutting-edge research. I have learnt several techniques in cell and molecular biology which I could
not possibly learn in my undergraduate degree. This has developed my confidence in research and
confirmed that academic research is the career path I wish to pursue after graduating.
References
Fukata, M., and Kaibuchi, K. (2001) Rho-family GTPases in cadherin-mediated cell-cell adhesion. Nat.
Rev. Mol. Cell Biol. 2(12) 887-897
Miao, H. Li, S. Hu, Y., Yuan, S. Zhao, Y., Chen, B.P.C, Puzon-Mclaughlin, W. Tarui, T. Shyy, J. Takada, Y.,
Usami, S., Chien, S. (2002) Differential regulation of Rho GTPases by 1 and 3 integrins: the role of
an extracellular domain of integrin in intracellular signalling. J. Cell Sci. 115(10) 2199-2206
Ory, S., Gasman, S. (2011) Rho GTPases and exocytosis: what are the molecular links? Seminars in
Cell and Developmental Biology. 22(1) 27-32
Reymond, N., Im, J.H., Garg, R., Vega, F.M., Borda dAgua, B., Riou, P., Cox, S., Valderrama, F.,
Muschel, R.J., Ridley, A.J. (2012) Cdc42 promotes transendothelial migration of cancer cells through
1 integrin. J. Cell Biol. 199, 653-668.
Reymond, N,, Borda d'gua, B,, Ridley, A,J. (2013) Crossing the endothelial barrier during metastasis.
Nat. Rev. Cancer 13, 858-870.
Tremont-Lukats, T.W., Bobustuc, G. Lagos, G.K. Lolas, K. Kyritsis, A., Puduvalli, V.K. (2003) Brain
metastasis from prostate carcinoma. Cancer. 98(2) 363-368
Weksler B.B, E.A. Subileau, N. Perriere, P. Charneau, K. Holloway, M. Leveque, H. Tricoire-Leignel, A.
Nicotra, S. Bourdoulous, P. Turowski, D.K. Male, F. Roux, J. Greenwood, I.A. Romero, P.O. Couraud
(2005) Bloodbrain barrier-specific properties of a human adult brain endothelial cell line. FASEB J.,
19, pp. 18721874
Isolation of secondary metabolites from Pseudomonas mesoacidophila

Student: Zainab Khan
Abstract: Secondary metabolites were isolated from a
Pseudomonas mesoacidophila fermentation broth grown in
two different types of media. The extracted metabolites were
purified by prep-TLC followed by prep-HPLC and studied
by UV, IR, MS, LA-ICP-MS and high resolution NMR
analysis. In addition, flash chromatography was also used to
purify two of the cell extracts from the PF broths; however
due to time restraints the structures of the isolated
metabolites were not elucidated.
Introduction: Antibiotic resistance has been on the rise for many
years and many important antibiotics have become inactive. In
recent years, creating analogues of existing antibiotics by chemical
modification has been the preferred method of drug development;
however, this approach has resulted in fewer viable antibiotics.1
More recently, due to advances in technology, natural products are
once again becoming an area of interest in drug discovery. 2 This
involves screening soil for bacteria which have evolved to produce a
diverse array of potent antimicrobial metabolites to kill their
competitors, such as simple phenylpyrrole derivatives like
pyrrolnitrin [Prn, 3-chloro-4-(2-nitro-3-chlorophenyl)-pyrrole, Fig
1]. Prn is a broad-spectrum antifungal compound that is active
against a wide range of fungi, yeast and Gram-positive bacteria.3 It is
an active component of PYRO-ACE and is one of the secondary
products of tryptophan metabolism.4
Figure 1: Structure of pyrrolnitrin

Aim: Initially, the aim of my project was to understand the
biosynthesis of the sulfated glycopeptide bulgecin in Pseudomonas
mesoacidophila.5 However, upon commencing my summer
placement the genome sequence of Ps. mesoacidophila became
available in our laboratory, revealing a Prn biosynthetic gene cluster.
This cluster seemed very interesting as previously Prn hasnt been
found in or isolated from this bacterial species. Thus from there
onwards the aim of my project became the isolation, purification and
characterisation of Prn, and other organic-soluble secondary
metabolites from the culture broths of Ps. mesoacidophila.
Materials and methods
Growth media
PS (Pseudomonas seed) medium: 1.5% w/v Nutrient Broth, 1%
glucose, pH 7.0
Supervisor: Dr Joel Loveridge

PF (Pseudomonas fermentation) medium: 3% w/v glycerol, 1.5%
Nutrient Broth, 0.1% tryptophan, 0.1% glucose, pH 7.0
PF (Pseudomonas fermentation) with KBr: 3% w/v glycerol, 1.5%
Nutrient Broth, 0.1% tryptophan, 0.1% glucose, 0.1% KBr, pH 7.0
BSM-G (Basal salts minimal growth media with glycerol): 0.324%
w/v K2HPO4, 0.1% NaH2PO4.H2O, 0.2% NH4Cl, 0.02%
MgSO4.7H2O, 0.05% tryptone, 0.05% yeast extract, 0.4% glycerol,
0.06% v/v trace elements solution, pH 7.0.
All media for bacterial growth were autoclaved at 121 oC for 15
minutes prior to use.
Extraction and purification of metabolites
A single colony of Ps. mesoacidophila was transferred to 100 mL PS
medium and grown at 28 oC, 150 rpm for 24 hrs. This seed culture
was then sub-cultured (1:50 dilution) into three 1 L portions of PF or
BSM-G medium, and grown at 28 oC, 150 rpm for 5 days. The
fermented broth was then pelleted at 6000 rpm at 4 oC for 20
minutes and the supernatant was separated from the bacterial cell
pellet. The pellet was suspended in acetone (200 mL), cells were
lysed by sonication, and the suspension filtered to remove any cell
debris. The acetone extract was concentrated under pressure; the oily
matter obtained was further extracted with DCM and concentrated
under pressure to yield a crude cell extract.
The supernatant was extracted with ethyl acetate and concentrated
under pressure to yield a crude supernatant extract. The crude cell
and supernatant extracts were then tested for antimicrobial activity
against E. coli JM109 and XL1-Blue by the disc diffusion assay
method. Plates were incubated at 37 oC and diameters of inhibition
zones were measured in mm. For purification of the secondary
metabolites, preparative TLC followed by prep-HPLC of the crude
cell extracts were performed. For prep-TLC, chloroform, ethyl
acetate and formic acid (5:4:1 v/v) was used as a solvent system, and
for prep-HPLC, the isolates of prep-TLC were eluted with a 0-100%
gradient of acetonitrile in water using a reverse phase C18 column.
For one of the PF broths supplemented with KBr, flash
chromatography was employed, the crude extract for this was eluted
with a 0-100% gradient of acetonitrile in water using a reverse phase
C18 column.
Findings:
- All of the crude cell and supernatant extracts prepared were
compared against a standard of pure Prn using thin-layer
chromatography (TLC; Rf 0.91); the results showed that none of the
crude extracts contained Prn.
- In addition, LA-ICP-MS was also carried out on the standard of
pure Prn (Fig 2). However, in general, ICP is a poor method of
detection for chlorometabolites as Cl has a high ionisation energy;
this can be seen from the small peaks observed in the pure Prn
sample even though the spot was concentrated. Negative results for
chlorometabolites are therefore not very informative.
- Moreover, the standard of pure Prn had a UV absorption band at

248 nm in chloroform.
Figure 4: ESI-MS of Pm1
Figure 2: LA-ICP-MS of pure Prn

- The crude cell and supernatant extracts of the first PF broth
prepared were tested for antimicrobial activity against E. coli JM109
and XL1-Blue using the disc diffusion assay method. The results
showed no clear inhibition zones for the crude supernatant extract;
however clear inhibition zones were observed for the crude cell
extract for both strains. As the concentration of the crude cell extract
was increased, the size of the clear inhibition zones increased (Fig
3). Subsequent work therefore focussed on the crude cell extract.
- The crude cell extract obtained from the first PF broth (PF1) was
purified by prep-TLC and all the different components were
separated. The spot at Rf 0.78 yielded an off-white solid (10 mg) and
this was further purified by prep-HPLC to yield an off-white solid
(Pm1; 8.6 mg).
- The mass of Pm1 was determined by electrospray mass
spectrometry to be 564 (Fig 4). The isotope ratio pointed towards a
compound containing two bromine atoms. LA-ICP-MS on a TLC
plate spotted with Pm1 confirmed the presence of bromine (Fig 5).
This finding was very interesting as terrestrial bacteria such as Ps.
mesoacidophila usually produce chlorinated metabolites, whereas
marine bacteria produce brominated compounds. Terrestrial bacteria
are usually able to synthesise the corresponding bromo-analogues
only when the culture medium is supplemented with bromide instead
of chloride.6
Figure 5: LA-ICP-MS of Pm1

- The 1H NMR spectrum of Pm1 (Fig 6) showed the presence of four
aromatic proton environments and a broad singlet peak at 4.66 ppm
which was due to protons exchangeable with the solvent (most likely
an amino group). The other peaks at around 2 ppm were due to
impurities such as water and acetonitrile. The 13C NMR (Fig 7)
showed the presence of 14 carbon environments. Moreover, the
results of the COSY, HSQC and HMBC were very hard to interpret,
due to the similarity between the chemical shifts for both 1H and 13C.
Figure 6: 1H NMR of Pm1 in CDCl3
Figure 3: Disc diffusion assay of 5, 10 and 20 L of PF crude cell

extract against E. coli XL1-Blue
Figure 7: 13C NMR of Pm1 in CDCl3
- For the BSM-G crude cell extract, purification was unsuccessful

and no metabolites were isolated.
- Another two PF crude cell extracts (PF2 and PF3) were also
prepared to isolate the spot at Rf 0.78; however this spot was not
present in the new crude cell extracts and the results of Pm1 were
not repeatable.
- A further two PF crude cell extracts were prepared but this time
one of the PF broths was supplemented with KBr to promote
production of bromo-metabolites, and both of these crude extracts
were purified by flash chromatography (Fig 8). Analysis of the
resulting fractions was not completed and TLC of both crude
extracts did not show the presence of Pm1.
Figure 8: Flash chromatogram of PF crude cell extract

supplemented with KBr
- A disc diffusion assay was also carried out on pure Prn against E.
coli JM109 and XL1-Blue and no antimicrobial activity was
observed.
- Furthermore, when the TLC plates of the crude cell and supernatant
extracts of both PF and BSM-G broths were visualised under UV,
blue/green fluorescent spots were observed; these are probably due
to siderophores (iron-sequestering compounds).7,8
- The fluorescence of the PF and BSM-G crude cell extracts was
therefore measured using a fluorimeter with excitation at 365 nm.
The fluorescence spectra confirmed that two different coloured
fluorescences were observed for PF with emission peaks at 440 and
465 nm. Whereas, only one coloured fluorescence was observed for
BSM-G crude cell extract with an emission peak at 445 nm (Fig 9).
known, the structure of purified isolates can be elucidated by NMR

and MS. Alternatively, bioautography could be performed on the
crude extracts by using developed TLC plates sprayed with a
suspension of fungal spores in broth, as a quick way of determining
which components of the extract show antifungal activity. In
addition, reverse transcriptase PCR could be performed on Ps.
mesoacidophila culture to determine whether the Prn gene cluster is
expressed under the experimental conditions used. Also, agar-based
growth media can be used to see if it has different effects to broths.
The value of the studentship to the student:
The summer studentship has been a very enjoyable experience and I
have relished every moment of it. It has significantly increased my
confidence in a research environment and I now feel more competent
at carrying out practical work. During my studentship, I was closely
supervised and taught by PhD student Gosia Kahl and my supervisor
Dr Joel Loveridge which was a great privilege, as I have now
acquired so many new skills and techniques beyond my chemistry
undergraduate studies. Overall, the studentship has been an excellent
experience and it has given me a valuable insight into what a career
in research entails on a daily basis. It has taught me that research is
all to do with patience, dedication and perseverance. Undertaking
this project has reassured me that a PhD in medicinal chemistry is
the correct path for me.
The value of the studentship to the lab (supervisors comment):
Zainab has done some excellent work in my laboratory this summer.
She worked hard, quickly learned the techniques required, and
managed her time well. At the end of her project, Zainab gave a very
good presentation to the Biological Chemistry group, and was able
to answer some quite challenging questions from the audience. She
will be co-author on at least one publication as a result of her work.
Furthermore, Zainab worked closely with my PhD student Gosia
Kahl, and I believe that both benefitted from this. I would strongly
recommend the Summer Vacation Scholarships to any
undergraduate student with an interest in research, and to any
academic with an interest in attracting excellent students to their
laboratory.
References
1.
2.
3.
4.
5.
Figure 9: Fluorescence spectra of BSM-G and PF crude cell extracts

6.
Future directions in which the project can be taken:

Crude cell extracts should be purified by flash chromatography and
the bioactivity of the resulting fractions should be tested against
fungi and both gram positive and negative bacteria. The genome of
Ps. mesoacidophila contains a number of putative biosynthetic genes
and gene clusters, including for non-ribosomal peptides, polyketides,
terpenes, butryolactones, lassopeptides, and bacteriocins, so a variety
of compounds should be produced. Once the antimicrobial activity is
7.
8.
Coates, A. R. M. & Hu, Y. Novel approaches to developing new

antibiotics for bacterial infections. Br. J. Pharmacol. 152, 114754
(2007).
Ling, L. L. et al. A new antibiotic kills pathogens without
detectable resistance. Nature 517, 455459 (2015).
Ligon, J. M. et al. Natural products with antifungal activity from
Pseudomonas biocontrol bacteria. Pest Manag. Sci. 56, 688695
(2000).
Van Pe, K. H. & Ligon, J. M. Biosynthesis of pyrrolnitrin and
other phenylpyrrole derivatives by bacteria. Nat. Prod. Rep. 17,
157164 (2000).
Imada, A., Kintaka, K., Nakao, M. & Shinagawa, S. Bulgecin, a
bacterial metabolite which in concert with .BETA.-lactam
antibiotics causes bulge formation. J. Antibiot. (Tokyo). 35, 1400
1403 (1982).
Van Pe, K. H. Biosynthesis of halogenated metabolites by
bacteria. Annu. Rev. Microbiol. 50, 37599 (1996).
Cody, Y. S. & Gross, D. C. Characterization of Pyoverdin(pss), the
Fluorescent Siderophore Produced by Pseudomonas syringae pv.
syringae. Appl. Environ. Microbiol. 53, 92834 (1987).
Bultreys, A., Gheysen, I., Maraite, H. & de Hoffmann, E.
Characterization of fluorescent and nonfluorescent peptide
siderophores produced by Pseudomonas syringae strains and their
potential use in strain identification. Appl. Environ. Microbiol. 67,
171827 (2001).

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Investigating Alzheimers disease in Down syndrome, using

transgenic mouse models

Introduction: Individuals with Down

assess whether the extra genes on

Aims: My project was aimed at

developed using a film processor

Figure 2: A plaque deposition in the Ts2Yey *J20 cross.

Ts3Yey x J20 cross: APP levels

Figure 3: Western blot

Statistical analysis of my Western blot

Figure 1: A deposition in hippocampus.

Figure 4: Levels of FL-APP relative to -actin.

The value of the studentship to the

Biochemical Society Studentship Report 2015

Structure-function relationship of the palmitoyl transferase DHHC5

Figure 1: Mitochondrial release of CoA via MPTP during

Figure 2: DHHC5 structure showing DHHC domain, region of

Assessment of results and outcomes of the studentship

Figure 3: Representative purification of cell

Figure 5: Relative palmitoylation of phospholemman in DHHC5 KO

Biochemical Society Summer Studentship Report 2015

TIE proteins: Bacterial ester domains

Biochemical Society Summer Studentship Report 2015

Biochemical Society Summer Studentship Report 2015

soluble fraction is analysed by SDS-PAGE to determine if protein is present, by comparing

Biochemical Society Summer Studentship Report 2015

Biochemical Society Summer Studentship Report 2015

Biochemical Society Summer Studentship Report 2015

the non-bond containing mutant protein versus the wildtype.

Biochemical Society Summer Studentship Report 2015

Figure 6: SDS-PAGE analysis of PnoED + PnET after 1 hour and 24 hours.

Biochemical Society Summer Studentship Report 2015

Biochemical Society Summer Studentship Report 2015

The Characterisation of Calcium Response and Neuron-Specific Markers on Different

Detection of Neuron-Specific Markers

Results and Discussion

Neural Progenitor Cells [P3]

Neural Progenitor Cells [P3]

de Groot, M.W., et al., Characterization of calcium responses and electrical activity in

Fig 2 53BP1 S366A and T670A mutations affect colocalisation of

Materials and Methods

Tissue cell culture, transfection, irradiation and inhibition of kinases

Results and Conclusions

Fig 3 CDKs do not phosphorylate S366 or

Fig 4 MAPK does not phosphorylate

Fig 5 CK2 does not phosphorylate S366. Hela

Departures from the Original Proposal

Characterising mutants of -phosphoglucomutase

Methods & Materials

Fig.4 Fluorine NMR spectra of K145A-AlF-G6P at pH 6.5

The 2D TROSY experiments of P138A-MgF3-G6P (Fig. 5A)

21, pp. 4195-4206, 2014.

Tables and Figures:

No Go Decay pathway nonfunctional.

Chaperone for proteasome 26S

Greatly reduced levels of

Complex binds stalled ribosomes,

Vacuolar protein degradation

Table 1: Knockout strains used in the latter experiments.

Figure 2. Maximum exponential growth

Biochemical Society Summer Vacation Studentship Report 2015

Background & Aims

Clone and express the truncated versions of HbGFP1

Spectroscopically analyse ensemble switching dynamics