CAPSTONE PERIOD 3



ENZBOT®
BY L AUREL J. C OOK
Pledge of Good Science..................................................................................4

Literature review ............................................................................................5

Rationale ......................................................................................................11

Limitations ....................................................................................................13

Materials ......................................................................................................14

Agar pre-poured Petri dishes ....................................................................................14

Cover glass .............................................................................................................14
Custom sequence ....................................................................................................14
desktop fan ............................................................................................................15
Dimmer heat lamp with bulb ....................................................................................15
Duct tape ................................................................................................................15
E. coli ....................................................................................................................16
Elemental Zinc powder ............................................................................................16
Glass aquarium .......................................................................................................16
Hemocytometer ......................................................................................................17
Heavy Plastic Sheet .................................................................................................17
Inoculation loops .....................................................................................................17
macbook pro and microscopy recording equipment ...................................................18
micro-Centrifuge ......................................................................................................18
Micro-centrifuge tubes ............................................................................................18
microscope .............................................................................................................18
Microscope slides ....................................................................................................19
Ninhydrin ..............................................................................................................19
Pipette ...................................................................................................................19
Plasmid recombination kit ........................................................................................19
Scissors ..................................................................................................................19
Thermometer (2x - celsius) ......................................................................................20
Thin Layer Chromatography Kit ................................................................................20
Wash bottle ............................................................................................................20
Water ....................................................................................................................20
Hypothesis, Variables, and Constants ..........................................................21
 Hypothesis ..............................................................................................................21
Independent Variable ..............................................................................................21
Dependent variable .................................................................................................21
Control groups ........................................................................................................21
Procedure .....................................................................................................22

Assembling the Incubator ........................................................................................22

Growing the E. Coli .................................................................................................24
Measuring the rate of growth ..................................................................................26
Calculating the rate of growth ..................................................................................28
Infecting the colonies ...............................................................................................29
Extracting the proteins .............................................................................................29
Tagging the amino acids ..........................................................................................30
Zinc-Dipeptide reaction ............................................................................................30
Citations .......................................................................................................32
 PLEDGE OF GOOD SCIENCE
I, Laurel Julia Cook, owner and founder of Enzbot®, aim to inspire a future of passionate
innovators with a brain for science and a heart for the Earth. In order to pursue this passion,
I hope that others will join me in this Pledge of good science, and that they, too, will
solemnly swear to;

A. Never use animals of all kinds in any way for any reason.

B. Always obtain materials from a sustainable source.

C. Never let greed drive our choices.

D. Always aim to make the wellbeing of others the top priority.

E. Never use radioactive material.

F. Always use recycled materials.

G. Never dump waste into the habitats of others.

H. Always be passionate about what we do.

I. Never let failure deter us from taking an alternative path to success.

Because I know that if we don’t make it our priority to take care of the Earth, then it may
not be able to take care of us.

Laurel Julia Cook, Founder of Enzbot® 2018

 LITERATURE REVIEW
The aim of Enzbot® was to originally design an enzyme that will react with botulinum “substrates” to produce a

visible reaction product. Upon reviewing the documents, however, it was then made clear multiple times that botox is not a

substrate, but rather a zinc-endopeptidase, which is an enzyme. Since a custom enzyme is not an option in the context of

dealing with botox, that idea was not going to work. In order to adapt to this, Enzbot® shifted to a more abstract concept

of designing protein complexes that consist of beta sheets which will be assembled into a cage. The amino acids that will

make up these beta sheets will include glutamine and arginine, respectively. This new approach has allowed for a more

reliable way of testing in the school laboratory, as well as reducing the need to test with botulinum toxins until it is

absolutely necessary. The cage proteins will surround a bioluminescent substrate, luciferin. The solution on the other hand,

will contain unrestrained luciferase, which is the enzyme that activates luciferin. The intention of this is so that when botox

serotype A cleaves the cage proteins open, the luciferase will be able to directly bind to the liberated luciferin. The glow

produced, however, can only be seen in the dark, which is why the final product will be packaged in recycled paper towel

rolls so the person performing this assay can use the paper towel roll to create a dark viewing space.

The Complete Amino Acid Sequence of the Clostridium Botulinum Type A Neurotoxin, Deduced by Nucleotide

Sequence Analysis of the Encoding Gene, written by Daphne E. Thompson et. Al. is potentially the most important

document in this entire literature review due to the fact that it not only contains the exact locations of certain enzymatic

activities listed in the amino acid sequence, but in that it also offers the complete amino acid sequence of the botulinum

toxin in FASTA format. [10]

To clear up some basic terminology before diving into this first article, it’s important to note that the sense strand is

the strand of double-stranded DNA that is identical to the mRNA structure, which ultimately winds up as the directly

corresponding codon translation strand for the protein. DNA is deoxyribonucleic acid, which is the chemical command

center for proteins that are produced in almost all living things. In order to understand the context of the project, it is crucial

that the processes of protein production are explained in full detail.

It is important to acknowledge that DNA is a double-helix composed of two strands that are joined together in a

particular order, as described by Watson and Crick. The parts of the DNA that are read by assembly proteins, called

enzymes, are called nucleotides, and they have special properties that make them only compatible for joining with the

corresponding nucleotide, just like a magnet can only attract the opposite charge of a second magnet. There are four

nucleotides, which are divided into two distinct groups; pyrimidines and purines. Purines can never form a bond with

purines; they must always form a bond with a pyrimidine or form no bond. (When a bond isn’t formed, it deactivates the

gene, and this particular type of deactivation of genes is the result of a particular type of mutation called a deletion

mutation.)

 The pyrimidine nucleotides of DNA include thymine and cytosine. The purine nucleotides of DNA are adenine and

guanine. With this information, it is logical to conclude that adenine always pairs with thymine and guanine always pairs

with cytosine. When looking at a nucleotide sequence, and there is either an A or a T, just assume it is paired with a T or an

A, but never with the same type of nucleotide base.

When a protein is needed in the body, the DNA is unable to directly produce a protein due to its’ size and role as the

universal template for the entire cell. Instead, a single stranded version of the particular gene that makes the protein, called

RNA, is synthesized. This synthesized RNA will then leave the nucleus and find a ribosome to start constructing the amino

acid chain (this is the protein) with the help of tRNA. The context of this project, however, will focus on the original sense

strand of the DNA and the amino acids that make up the protein.

The first article mentions several methods that they used to determine the complete amino acid sequence of the

BoNT A protein, where the first of which was extracting the DNA from the botulinum type A culture that was grown in a

substance called agarose that followed the initial extraction method of the DNA from C. Botulinum. First and foremost, the

DNA was radio-labeled before a southern blot analysis took place. [10] The radio labeling of DNA involves using particular

enzymes, called restriction enzymes, to bind to the DNA at particular patterns in the DNA that mark the ending of one gene

and the beginning of another. The TATA box that was described earlier is that particular place, for the molecular properties

of Adenine and Thymine are slightly different than that of Cytosine and Guanine.

Once the genes are radio-labeled, the southern blot analysis can take place. [10] The purpose of a southern blot

analysis is to see if a particular gene is located on the samples of DNA that were obtained from the extraction process.

(Marmur extraction.) The first step in the process of southern blot analysis is to cleave the DNA with enzymes. The purpose

of cleaving the DNA into two separate strands is so that the radio labeled gene corresponds to the complement, and forms

a double strand of DNA again. (Again, this process requires the use of enzymes, for any chemical bonding process requires

energy donors to initiate the process.)

Once the DNA has been cleaved into two separate fragments, it can then undergo gel electrophoresis, [10] which is

a very intricate process involving the separation of DNA based on density. Electrophoresis begins with restriction enzymes

that cleave DNA at gene endpoints. (Discussed above.) It’s important to separate the genes so that when the genetic

material is placed in the gel for the electrophoresis, it will filter out by size due to the size and charge of the molecules. The

larger genes will fall down the wells towards the bottom of the gel, while the smaller genes will remain on the surface layer.

It takes about twelve hours for the genes to sort through the filter based on size, and when that’s done, the gel containing

the genetic information will be transferred to a filter so that when it is analyzed, the observer won’t accidentally mix up the

separated strands on accident; one small bump is enough to mess up a process that took all night to complete, and will

most likely cause a great degree of annoyance and distress if this is to be done in a timely manner.

After transferring the DNA fragments to the filter, the next step is to expose the genes to the radio-labeled DNA

template to attract the corresponding gene. This is not visible to the naked eye, so an X-ray film is required to visualize
where the radio-labeled DNA probe went. Unfortunately for some cases, the X-ray will reveal that the corresponding gene

is not located in the sample, and the scientist will have to start all over from scratch. [9]

In The Complete Amino Acid Sequence of the Clostridium Botulinum Type A Neurotoxin, Deduced by Nucleotide

Sequence Analysis of the Encoding Gene, there were a lot of other procedures that followed the southern blot analysis. The

gene was successfully found in the sample, so the next step involved mapping where the gene was located in the strand

with the corresponding restriction enzymes. Below is the image from the document that shows the genomic map that the

researchers have created in correspondence to the genes that successfully maps the BoNT-A gene.

Upon mapping out the location of the gene, it was then stated that the gene is a 150-kDa fragment. kDa means kilo-

daltons, and a dalton is the weight of one hydrogen atom, so this is basically 150,000 daltons in weight. Due to the risks

involved in accidentally creating toxic molecules, the scientists purified the sample and hydrolyzed it to separate the

sequences coding for the light chain from the genes coding for the central portion and the latter, which was separating the

genes coding for the heavy chain from those genes that code for the central portion.

In order to produce a protein from a DNA sample, there must be a way to read the DNA sequence, create

corresponding RNA, and then transport that RNA to a ribosome to then ‘print’ that protein. Finding out the multiple ways

that scientists produce protein molecules has been a very long but very enjoyable journey, and that brings in a second

article to compare.

Phages as Tools for Functional Nanomaterials Development by W-J Chung, M Sena, A Merzlyak, and S-W Lee, who

are all from the University of California at Berkeley, is probably the second most important article in this entire review, for it

serves to provide an application that can be used in the production of Enzbot. The article is more explanatory than

anything, for it isn’t a research paper, but rather a general textbook article that provides examples of different applications
for phage printing. It may not be ideal, but the descriptive examples will serve major volumes in this project, so it is very

important that this article is included in this review.

The main focus of the article Phages as Tools for Functional Nanomaterials Development is centered around M-13

bacteriophages that are used in laboratory settings to create things that people cannot create by hand. Proteins, for

example, can only be manufactured by living cells on the molecular level, so scientists have to get creative when they want

to produce a protein on command.

The most common method of creating a protein in a laboratory is performed with the exploitation of viruses. A virus

is pretty much a tiny flash drive; it pretty much tells the cell to print the DNA that is encapsulated within the virus by forcing

the DNA into the cytoplasm, which causes enzymes to begin prepping the DNA to be converted to RNA and finally print

proteins that the DNA encoded. The proteins that a virus typically encode for are just more virus proteins that will simply be

bound to each other to create a new virus and cause cell lysis to the cell so that the new viruses can be dispersed to

surrounding cells.

This natural exploitation of the ribosomes in cells to produce proteins from a completely foreign strand of genetic

information is often used in laboratories by scientists. M-13, a bacteriophage that infects E. Coli, is the most commonly

used virus by many types of scientists, from pharmacologists who use it to print the many enzymes that are found in drugs,

to computer engineers who use the viruses inside of lithium-ion batteries simply for the structural similarity to copper

nanowire.

The first article mentioned the use of M-13 bacteriophages to print the three major pieces of the botulinum toxins

(light chain, heavy chain, and central portion) to study the toxins without the toxigenic effects present. It was deduced from

the study in the first article that as a result of testing the three components, the zinc ion (Zn2+) resided in the heavy chain

of the molecule, and was responsible for the enzyme’s high affinity for the synaptobrevin associated protein (SNAP-25).

Synaptobrevin associated proteins are proteins that are found on the ends of axons that allow for the release of

neurotransmitters from one neuron to another. Neurotransmitters are the molecules that deliver the electrical impulses to

each neuron, where dendrites receive the electrical impulses from the axon terminals of the other neuron. Botulinum

neurotoxins will bind and cleave SNAP-25, which will ultimately inhibit the release of acetylcholine, which is the

neurotransmitter that is associated with muscular contractions that grant mammals the ability to move.

The effect that BoNT type A has on the SNAP-25 and SNARE proteins is described in a third article, The Destructive

Effect of Botulinum Neurotoxins on the SNARE Protein- SNAP-25 and Synaptic Membrane Fusion, by Bin Lu and edited by

Vladimir Uversky, all of whom are from the Center for Membrane Biology at the University of Virginia in Charlottesville,

Virginia.

One of the most important parts of the article is located on page two, where Lu mentions, “BoNT/A cleaves nine

amino acids from the C-terminus of SNAP-25 between Gln197 and Arg198,…”(Lu 2). With this said, it becomes much more
apparent as to where exactly the botulinum neurotoxin type A binds to, thus allowing the focus to shift to protein cages

composed of glutamine and arginine dipeptide chains.

The study that the author employed included the pulldown method so they could study the arrangement of the

protein in space, or better known as the conformation. Similar methods to the pulldown method are very helpful, and they

are probably going to be the most employed method by Enzbot®. Another article, A Guide to Simple, Direct, and

Quantitative In-Vitro Binding Assays, by Stefanie Lapetina and Hava Gil-Henn, happened to mention that the pulldown

method is entirely in-vitro. In vitro is latin for “in the glass,” which denotes the fact that in-vitro procedures take place

outside of bodies and can be performed without the use of biological organisms, which is really good considering the fact

that this procedure doesn’t contribute to animal testing of any kind.

Animal testing is a major ethical concern in science, and Enzbot® aims to put it to a stop in our procedures. One

major article that touches on the issues with using mice as a Botulinum assay method makes several important statements

addressing the need for better methods. Detection of Botulinum Neurotoxin Serotype A, B, and F Proteolytic Activity in

Complex Matrices with Picomolar to Femtomolar Sensitivity, by Dunning, F. M., Ruge, D. R., Piazza, T. M., Stanker, L. H.,

Zeytin, F. N., & Tucker, W. C, starts off with mentioning the fact that methods for detecting the presence of Botulinum toxins

in food are needed in order to ensure public safety. According to the paper, “In all botulism cases, whether naturally

occurring or intentional, early diagnosis of intoxication is critical for effective treatment and identification of the source of

the toxin.” [4]. With this statement serving like the Bat Signal, it is clear that Enzbot® needs to make a highly effective

assay method protocol along with the design of the product so that it can be used by just about anyone.

The people behind this fourth article have created their own in-vitro assay technique for detecting Botulinum toxins,

which is stated as; “These assays use fluorogenic protein reporters consisting of an N-terminal cyan fluorescent protein

(CFP) moiety and a C-terminal yellow fluorescent protein derivative Venus moiety linked by a BoNT substrate, residues 141

to 206 of SNAP25 in BoTest A/E and residues 33 to 94 of synaptobrevin in BoTest B/D/F/G. In the absence of cleavage,

excitation of CFP results in Förster resonance energy transfer (FRET) to Venus, quenching CFP emission and exciting

Venus emission. Upon linker cleavage by BoNT, CFP and Venus become separated, pre- venting FRET; CFP emission

increases, and Venus emission de- creases. The ratio of these two emissions gives a quantitative mea- sure of BoNT

activity. Furthermore, as long as the BoNT remains active, the BoTest reporters will continue to be cut; thus, the assay can

be read until the desired sensitivity is achieved.” [4]


The difference between this method and the method that Enzbot® will use is that unlike the complex chain of

reactions going on in this product, Enzbot® will use a simplistic design of a luciferin substrate encapsulated inside of a

custom SNAP-25-derived cage. The goal of this is so that when BoNT/A cleaves at the Gln-Arg residues on the protein

cage, the luciferin will be released into the medium in which the solution of enzymes, luciferase, will be suspended. Upon

liberation, the luciferin should bind to the active site of the luciferase, which will produce the same chemical reaction that
can be observed in the abdomens of fireflies. The experimental design for how this is going to be pulled off, however, will

come later in the project.

Diving back into the concerns raised by animal testing, the article addresses the need for new methods by stating,

“Alternative, animal-free, standardizable assays for the detection of BoNT activity with mouse bioassay sensitivity are a

critical need for diagnostic, environmental, biodefences, and pharmaceutical testing.” [4]. This statement not only applies

to the detection of botulism in a living organ system, but it also applies to the ability to test the product without using entire

organisms in the process.

This final article, Organs-on-a-chip: Current applications and consideration points for in vitro ADME-Tox studies by

Seiichi Ishida from the Division of Pharmacology at the National Institute of Health Sciences in Japan, provides a genius

approach to testing the product so that the effects on the human body can be studied in a very high-tech manner that

reduces the need for using whole animal models. [11]

The way these organs on a chip work is quite simple, really. The chip is comprised of sterile plastic that contains

several cavities that represent organs in the body. These cavities are lined with cells that are grown in labs that can be

found in the particular organ, so for example, let’s say one of the organs on the chip happened to represent the stomach;

the cavity for the stomach would be lined with mucus, contain hydrochloric acid, and it would also contain cells from the

stomach. [11]

Cavities that make up the organs are connected via channels that correspond with those that are found in the body

that pump blood. The arteries are represented by a channel that pumps oxygen-rich fluid that simulates blood, while the

veins are represented by deoxygenated fluid that tends to make its way back to the lungs. [11]

The whole organ chip can be powered with a simple Arduino circuit board, [11] which makes the whole thing even

easier for innovators to employ this method. Enzbot® looks forward to employing this method, as well as other methods in

the experimental phase, which is the next portion of this paper.

 RATIONALE
The overall aim of Enzbot® is to create a liquid solution that will allow just about
anyone to enjoy their food without the fear of getting botulism. If everything goes as
planned, then the final product should create a visible glow when viewed in the dark. (It can
be viewed in the dark by using an up-cycled cardboard tube from a paper towel roll.)

The experimenting and designing stages are going to be happening simultaneously. As

of right now, the current idea for the final design is a cage-shaped protein that can be
“unlocked” by botulinum toxins. Botulinum toxin type A cleaves Synaptobrevin-Associated
Protein-25b at a peptide bond between amino acids 197 and 198. These two amino acids are
glutamine and arginine, respectively.(*1,*2) The active ion in botulinum neurotoxin type A
is a zinc2+ ion, which is the part of the molecule that has the high affinity for the dipeptide
197-198.

A great place to start off with this project is to test how zinc will attract various
molecules consisting of the glutamine-arginine bonds. If the zinc forms a bond with the
dipeptides, then it will be much easier to plan out how to build a protein cage that will keep
this affinity in mind, since working with botox might not be the safest option just yet. It
may seem a bit abstract, but the whole design of this experiment process is to verify things
are working before throwing in larger and more complicated strategies.

*1(Thompson, D. E., Brehm, J. K., Oultram, J. D., Swinfield, T., Shone, C. C., Atkinson, T., . . . Minton, N. P. (1990). The Complete Amino Acid Sequence of the Clostridium botulinum
Type A Neurotoxin Deduced by Nucleotide Sequence Analysis of the Encoding Gene. Eur. J. Biochem,189(73-81), 1990th ser., 73-81. Retrieved April 2, 2018.)

*2 (Baldwin, M.R., Bradshaw, M., Johnson, E.A., & Barbier, J.T. (2004, September.). The C-Terminus of Botulinum Neurotoxin Type A Light Chain Contributes to Solubility, Catalysis, and Stability.
Retrieved April 20 from https://www.ncbi.nlm.nih.gov/pubmed/15294297)
 LIMITATIONS
Enzbot®, along with any innovative science project, will face major limitations.
Probably the largest limitation of this project is the fact that there will be no true way of
knowing that everything is being created correctly on the molecular level, being that there is
no way to see atoms without spending millions on expensive lab equipment. We can,
however, use mathematics to overcome the main barrier of being unable to know exactly
how everything is falling into shape. One such mathematical strategy would be the use of
differential calculus to predict when the amount of protein being produced by an e-coli
colony is at the optimal level for experimentation, or by using stoichiometry to understand
just what exactly is being produced as a byproduct of any reaction that occurs in the in-vitro
analyses that will take place.

Another issue is the fact that botulinum toxins are not going to be easy to obtain. Only
licensed pharmacologists may purchase Botox® online, so it’s impossible for a high school
student to simply buy a vial online. Botulinum toxins are also the deadliest substance
known to mankind, so it’s going to be a bit risky if Enzbot® ends up testing with the toxins
in any school laboratories. Luckily, there are ways to go about this issue. The first, and
probably the easiest method, is to use computer programs that can predict molecular
interactions using real data collected from thousands of experiments that have been
published in scientific journals. Another way to go about this is to partner with someone
out there who can actually work with the botulinum toxins; there are almost 8 billion
people on the planet, after all - there has to be someone who has the experience required to
safely work with botulinum toxins.

Finally, the last, and probably the most obvious limitation, is funding. Money is a
difficult thing to obtain, especially as a young scientist without any experience in financial
situations. This is where Enzbot® has to get creative in its ways of fundraising, such as
implementing completely bizarre fundraising events like pizza eating contests, raffles (Raffle
tickets would be a nickel each), and probably the most unique of them all would be a Lego
fire walk. The last idea is a bit bold, but there are some interesting things that people would
actually pay money for. If that doesn’t work, then there’s always the option of even putting
on a “chemistry show” for people for $1 per person where some really cool reactions could
be demonstrated for a younger audience using simple things like dry ice and things like that.
It’s popular among kids.
 MATERIALS
AGAR PRE-POURED PETRI DISHES

Agar will serve as the growth medium for the E. coli that are being grown in this
experiment. Agar is a gelatinous mixture of agarose, a natural protein that is found on
seaweed, and water. It resembles jello, and it can have just about any substance mixed into
it, from pig blood to the shells of yeast. In this laboratory procedure, the agar should contain
yeast extract, which can be lightly sprinkled onto the pre-poured agar plates if necessary.

Agar pre-poured petri dishes will be obtained from Amazon for a price of about 20
USD. Free shipping is available for Amazon Prime members, so that’s a plus. This set comes
with

COVER GL ASS

Cover glass is a great way to protect the contents of a microscope slide from the lens of
a microscope objective lens. It’s made of 0.1mm thick glass, so it’s very fragile and easy to
break if the stage is raised too high. In this experiment, cover glass will be carefully used to
protect the e-coli from any dust that may be floating around the microscope stage.

At Enzbot® Headquarters (my house), there is a plentiful supply of cover glass, so

there is no need to buy more.

CUSTOM SEQUENCE

The goal of this initial experiment is to make sure that zinc still has a high affinity for
residues 197 and 198 even when they are isolated from the SNAP-25 protein. The custom
sequence is going to be a simple gene for a dipeptide consisting of the same glutaminyl-
arginine bond. The website where this can be ordered from sells the custom sequences for
only eleven cents per base pair - this will cost less than a dollar, with the exception of the
few protective codons. (TATA boxes.)
DESKTOP FAN

The desktop fan serves as a temperature-control for the e-coli that is being observed under
the microscope. Since E. Coli should be kept from 28 degrees Celsius to 45 degrees Celsius,
it is important to make sure that there is a way to cool it off if the light below the stage
creates too much warm air. The fan will simply blow the hot air away, thus reducing the
temperature of the specimens on the slide.

D I M M E R H E AT L A M P W I T H B U L B

A dimmable heat lamp will be purchased from Amazon for 24.43 USD to use as the heat
source for the incubator. The lamp has built in clips so it can clip onto the aquarium without
having to install any extra fixtures. It also comes with a switch that can be used to control
the brightness of the incandescent light bulb, so the optimal temperature can be reached.
Zoo Med’s Day/Night Light Combo Pack will be purchased for 11.47 USD and the day light
will be used in the Zoo Med Deluxe Dimmable Clamp Lamp with 8.5-Inch Dome on the
incubator.

Lindsay, H. (2017, April 24). How to Make an Incubator to Grow Bacteria. Retrieved June 8, 2018,
from https://sciencing.com/make-incubator-grow-bacteria-7908191.html

D U C T TA P E

Duck Brand 1017800 Advanced Strength Duct Tape will be purchased from Amazon for 5.99
USD. Duct tape will be used to hold the thermometer to the side of the incubator, as well as
to fix the plastic cover to the top of the aquarium.

Lindsay, H. (2017, April 24). How to Make an Incubator to Grow Bacteria. Retrieved June 8, 2018,
from https://sciencing.com/make-incubator-grow-bacteria-7908191.html
E. COLI

E. Coli is probably the second most used bacterium in laboratories, with yeast probably
being the most common. The reason why E. coli is being used in this lab instead of yeast is
because M13 bacteriophages are the most abundant viruses on the planet, so it will be
extremely cheap to get them for the project. M13 phages will be used to infect the E. coli so
that the E. Coli will print any custom genetic sequence in the form of a polypeptide.

E L E M E N TA L Z I N C P O W D E R

Elemental zinc powder is probably the purest form of zinc available in powdered form.
It’s important to note that there is no such thing as a pure substance in nature, and purity is
simply impossible to find in any particular substance. It is, however, helpful if a substance
can be obtained in its purest form. Elemental zinc powder will be obtained over Amazon,
where it is sold for a pound (or 453.592 grams.). It is abut 10 USD for a pound of zinc.

GL ASS AQUARIUM

A 10 gallon glass aquarium tank will be used to serve as the main compartment of the
incubator. The aquarium dimensions are 22”x12”x13.5”, and it can be purchased from
Walmart for 14.72 USD. Glass is very heat-resistant, so that’s why glass is being used
instead of plastic.

Lindsay, H. (2017, April 24). How to Make an Incubator to Grow Bacteria. Retrieved June 8,
2018, from https://sciencing.com/make-incubator-grow-bacteria-7908191.html
HEMOCYTOMETER

Hemocytometers are microscopic grids that are printed onto cover glass to count blood
cells. The grid squares help to keep track of where the cells are when it is time to count
them, since it’s easy to lose track if the cells are not confined to cells. The way this works is
that a camera will photograph the cells at any given time, and the photo will be analyzed.
The cells will be confined to a grid system, and any cells that are on the outer grid lines are
immediately scratched out with a marker (Or with an online photo editing tool.). Then, the
cells are counted going square by square until the entire area of the grid has been counted.
Then, the following equation is used to calculate how many cells are in a mL of the sample;

Total cells/mL = Total cells counted x dilution factor x 10,000 cells/mL

# of squares

Hemocytometers are used for just about any kind of cell, whether it’s blood cells, or in the
case of this experiment, e. coli cells.

Grigoryev, Y. (2015, November 30). Cell Counting with a Hemocytometer: Easy as 1, 2, 3. Retrieved
June 7, 2018, from https://bitesizebio.com/13687/cell-counting-with-a-hemocytometer-easy-as-1-2-3/

H E AV Y P L A S T I C S H E E T

A heavy 12”x24”x1/8” acrylic plastic sheet will be purchased from McMaster-Carr for 14.27
USD to help keep the heat inside of the incubator. Acrylic plastic will melt at about 160
degrees Celsius, so there’s no danger of it melting while being used on a low temperature
incubator.

Lindsay, H. (2017, April 24). How to Make an Incubator to Grow Bacteria. Retrieved June 8,
2018, from https://sciencing.com/make-incubator-grow-bacteria-7908191.html

I N O C U L AT I O N L O O P S

Inoculation loops are loops of wire that sort of resemble a bubble wand. They are used for
streaking bacteria on an agar plate in patterns so that the colonies can be identified. These
are relatively cheap, with an 8” holder and 5x 2.2” reusable Nichrome wire tips being sold
on Amazon for 14.29 USD.
MACBOOK PRO AND MICROSCOPY RECORDING
EQUIPMENT

A MacBook Pro will be used to obtain real-time data from the microscopy imaging. The
microscope at Enzbot® Headquarters has a built in camera at the top of the neck, as well as
an adapter to plug it into the USB to USB-C adapter, which will ultimately end up
connecting to the Macbook Pro™. The camera is an AmScope® MU1000 10MP Digital
Microscope Camera, which is compatible with Mac OS X software. The program used will
be AmScopeX for Mac™.

MICRO-CENTRIFUGE

A micro-centrifuge will be used to separate the dipeptides from the e. coli specimens upon
cell lysis. Micro-centrifuges will be used at Fairchild Wheeler in the biotech lab on the third
floor, since Enzbot® Headquarters does not own a centrifuge. Centrifugation is a process
that separates fluids based on density by using centripetal force to move items of a greater
density outwards, while items of a smaller density remain towards the center. Tubes are
placed on their sides so when they are picked up, everything remains in their respective
layers.

MICRO-CENTRIFUGE TUBES

Micro-centrifuge tubes be used to contain the samples of lysed e. coli bacteria and the
proteins that will be separated. These can be purchased from just about any online science
supply retailer, but Fairchild Wheeler has boxes of micro-centrifuge tubes that fit the
centrifuges here.

MICROSCOPE

The microscope will allow for the viewing of the e. coli on the hemocytometer for counting
so that the growth rate can be observed. Objective lenses ranging from 4x, 10x, 100x, and
400x are all available for use along with the microscope at Enzbot® Headquarters.
MICROSCOPE SLIDES

As with any microscope session, slides are needed to hold the specimens. Small glass
microscope slides are located at Enzbot® Headquarters to be used in the viewing of the
specimens.

NINHYDRIN

Ninhydrin is a compound that is used to stain amino acids, and will produce a range of
colors depending on which amino acids it binds to. In order to observe this, however, it is
important that a TLC kit (Thin layer chromatography) is used to properly view the
polypeptide samples on the silica gel.

PIPETTE

Pipettes are needed to add small amounts of a substance to a solution. The pipettes will be
used for many things, from adding the ninhydrin to the strips of polypeptides, to the
addition of the zinc solution to the dipeptide suspension.

P L A S M I D R E C O M B I N AT I O N K I T

A plasmid recombination kit not only provides detailed instructions for how to add a gene
to an M13 phage plasmid, but it will also provide the materials needed to infect the E. Coli
bacteria. This kit is about 250 USD but it’s also the most important part because it provides
for the method of producing the desired dipeptides for analysis.

SCISSORS

Scissors will be used to cut the duct tape so that it can be used in the construction of the
incubator. There’s really not much to say about scissors other than the fact that running
with them is ill-advised.
THERMOMETER (2X - CELSIUS)

Thermometers will come in handy for two parts of this initial experiment. The first part is
the construction of the incubator, in which the thermometer will be placed up to the side of
the tank on the inside so that the temperature can be monitored. The second use will be for
the monitoring of the bacteria when it is placed on the microscope slides so that it remains
at the same temperature as it was in the incubator.

T H I N L AY E R C H R O M A T O G R A P H Y K I T

Thin layer chromatography kits come with the silica gel and reagents needed to screen for
the presence of particular amino acids in a substance. The kit costs 200 USD but it is also a
very important assay kit that will provide evidence of the presence of the glutaminyl-
arginine dipeptides in the final substance.

WA S H B O T T L E

Wash bottles are important in laboratories because they allow for the removal of debris by
using high pressure to propel water out of a narrow tube. This is akin to a mini power
washing hose, which is used to clear debris from just about anything.

WAT E R

Water is needed in just about every solution being made in this experiment. Water can act as
a coolant, a method of removing debris, and as a solvent.
 H Y P O T H E S I S , VA R I A B L E S , A N D C O N S T A N T S

HYPOTHESIS

If various concentrations of stained Glutaminyl-Arginine dipeptides are added to a

suspension of 10 mL of water and 100 mg of elemental zinc powder, then the zinc will pull
the dipeptides towards it.

I N D E P E N D E N T VA R I A B L E

The independent variable, or the variable that is changed, is the Concentration of the
Glutaminyl-Arginine dipeptides (g/mL) that is being added to the solution of elemental
zinc.

D E P E N D E N T VA R I A B L E

The dependent variable, or the variable that is changing with respect to the independent
variable, is the size of the clusters of Glutaminyl-Arginine dipeptides that have been drawn
to the zinc.

CONTROL GROUPS

The control in this experiment will be the proteins of lysed bacteria without the
recombinant plasmid proteins present. (QR).
 PROCEDURE

A S S E M B L I N G T H E I N C U B AT O R

The first step in this experiment is to construct the incubator that will be used for
incubating the Escherichia coli cultures. Here are the materials that will be used from the list;

• Dimmer heat lamp and bulb

• Duct tape

• Glass aquarium

• Scissors

• Thermometer

• Thick Acrylic plastic cover

Some other supplies that will be needed include;

• Alcohol prep wipes

• Electrical outlet

• Latex gloves

• Microfiber cloth

• Window cleaning solution (Windex®™ kills most bacteria.)

Before assembling the incubator, it is important to put some latex gloves on so finger
prints don’t get on the glass. Finger prints contain many species of bacteria which can wind
up in the petri dishes. Wipe down the surfaces of the interior of the aquarium so that any
pre-existing bacteria growing inside is gone. Window cleaning solution will be used to wipe
down the interior of the tank with the microfiber cloth. The aquarium should then be placed
on its side to dry out so there is no windex still in there. The Windex® should be used
sparingly since it will take less time to dry if less Windex® used.
 In the meantime, while the glass aquarium is drying, an alcohol prep wipe will be
removed from the little package and used to wipe down all surfaces of the piece of thick
acrylic plastic to try to sterilize it as much as possible. This acrylic plastic is going to be the
surface that the petri dishes will be on, so it’s important for this to be the most sanitary part
of the incubator.

The acrylic plastic should dry very quickly, but if the aquarium may be wet. The
aquarium should be allowed to dry completely before advancing to the next step.

Once the aquarium is completely dry looking, the thermometer should be placed flush
against the glass so that it can be viewed from outside. It should be placed with the bulb
facing the opening of the aquarium since it will be inverted later. The thermometer will then
be secured to the side of the aquarium with a piece of duct tape so that it remains in place.
It should be noted that the thermometer should not touch the bottom of the aquarium.

Upon placing the thermometer inside of the aquarium, the next step is to screw in the
lightbulb into the heating lamp. Once the lamp is finished, the aquarium will be inverted
and placed on top of the acrylic plastic, while the lamp is placed on the top of the glass. The
final product should look like this;

Source of instructions: Lindsay, H. (2017, April 24). How to Make an Incubator to Grow Bacteria.
Retrieved June 8, 2018, from https://sciencing.com/make-incubator-grow-bacteria-7908191.html

GROWING THE E. COLI

The next step is to grow the E. coli in the incubator that was just constructed. Before
this can happen, however, the pre-poured agar plates must be inspected immediately upon
delivery because sometimes errors happen in the shipping process that end up
contaminating the agar plates and causing bacteria to already be present upon receiving the
agar. It’s important to note that agar plates must always be stored upside down so
condensation droplets do not end up forming in the top of the dish. When the droplets get
too large, they will fall onto the bacterial colonies, causing them to dissipate into the
surrounding colonies, thus disrupting the genetic equilibrium among the micro colonies.

If all ten petri dishes have arrived intact and without a trace of bacteria growing inside,
then the experiment may continue as planned. Here is the list of the materials that will be
used in this section of the experiment;

• E. coli culture (Obtained from Carolina® Biological Supply Company)

• Pre-poured agar petri dishes

• Incubator

• Inoculation loop

Some other supplies that should come in handy include;

• Safety goggles - (It’s the law) Prevents E. coli or broken glass from getting in the eyes.

• Sterile nitrile/latex gloves - Keeps hand bacteria away from the petri dishes.

• Lab coat - Keeps skin and cells from shedding and landing into the petri dishes.

• Bouffant (A shower cap works excellently.) - Prevents hair or yeast from hair from falling
and getting into the bacterial cultures.

• Laboratory mask - Protects the bacteria from foreign mouth and nasal bacteria.

• Open flame source (bunsen burner or alcohol burner)

• Masking tape

• Permanent marker
 The first step upon affirming that the petri dishes are not contaminated involves wiping
down the lab surface, careful washing of the hands, and putting on safety goggles, a lab coat,
a shower cap/bouffant, a face mask, and gloves.

The burner will then be lit and an inoculation loop will then be placed in the flame for
merely a millisecond; it doesn’t have to be exactly a millisecond, but it should be done in
such a quick manner to avoid charring the metal. If the metal chars, a paper towel will come
in handy to remove the ash from the loop. If this inoculation loop is made of plastic, then a
cotton ball dipped in rubbing alcohol should do the trick for sterilizing the loop. Never
store alcohol next to an open flame; alcohol is highly flammable.

Once the inoculation loop is sterile, the lid from the agar plate will be lifted and put
aside, with the inside facing upwards. Efforts will be made to avoid touching the inside of
the petri dish. The loop will then be dipped into the e. coli specimen tube and very
delicately streaked across the surface agar with the loop without scratching the surface. The
majority of the streaks will be made in the first quadrant of the petri dish, and gradually
decreased to the fourth quadrant, respectively. The agar should not be broken or scratched
while doing this; if so, it’s fine, but it may cause the E. coli to get under the agar and
separate it from the dish, causing it to fall while being stored upside down.

After each petri dish has been inoculated, the bottom of the petri dishes (the lid part)
should be clearly labeled with a piece of masking tape and a permanent marker. This is
important so that these samples can easily be identified from the other samples. After this,
the petri dishes will be placed upside down (agar up up, lid down on the bottom.) on the
acrylic plastic so that the aquarium can then be placed on top of the plastic, enclosing the
petri dishes inside. Before the aquarium is placed on top, however, it is important to make
sure that the light is set to a brightness that will allow the inside of the aquarium to be
somewhere around 37 degrees celsius, which is the optimal temperature for e. coli growth.

Once the desired temperature (37 degrees celsius, +/- some) is reached, the e. coli can
then be left to incubate inside of this makeshift incubator for the amount of time it takes for
the desired colony numbers to be reached. This can be determined by using some
differential calculus to create an accurate idea of the rate of growth over time.
M E A S U R I N G T H E R AT E O F G R O W T H

In this section of the procedure, the cells in each E. Coli colony will be counted in 1
hour intervals using a hemocytometer to approximate how many cells per mL there are. A
standard petri dish should contain around a mL of bacteria at a given time, so this
approximation should do the trick in estimating the size of the population.

Several supplies will be needed to perform this test;

• AmScopeX camera

• AmScopeX for Mac™

• Cover glass

• Desktop fan

• Duct tape

• E. Coli colonies

• Hemocytometer

• Inoculation loop

• MacBook Pro

• Microscope

• Microscope slides

• Scissors

• Thermometer

• Wash bottle

• Water

Other supplies:

• Alcohol prep wipe

 The first step will involve taping the thermometer to the diaphragm of the microscope
so that the bulb is over the viewing area. The thermometer should be monitored every few
minutes to make sure it isn’t exceeding 37 degrees celsius or else it could alter the results of
the procedure.

Once the thermometer is in place, the inoculation loop should be sterilized by using an
alcohol prep wipe. The inoculation loop will then be left to dry while the first agar petri dish
is removed from the incubator. With the inoculation loop, the most dense colony on the
plate should be lightly touched so it isn’t disturbed too much, before it is then wiped onto
the slide and spread evenly in the center of the slide. Finally, the cover glass should be
placed on top of the sample, while the slide with the hemocytometer should then be placed
on top of that. A note should be made about which culture this is before it gets placed back
into the incubator.

Once the slide has been prepared, it will then be placed onto the viewing stage of the
microscope. The camera will then be connected to the macbook pro using the proper cables
to do so. Then, AmScopeX™ for Mac™ should then be opened, and instructions for how to
load the image onto the computer screen should be followed. (It differs every time due to
updates.)

Once the image is loaded, the stage will then be adjusted along with the objective
lenses until the hemocytometer is in full view, and the bacterial specimens are visible. E.
Coli should be a rod shaped bacterium; if there are other bacterial species present, a note
will be made, and they will be disregarded in the counting of the E. Coli specimens.

After making the image as clear as possible, a screen recording will be taken to film the
bacteria for one hour. After the hour is done, the screen recording will be opened and the
scrub bar at the bottom should then be brought to the initial time that the screen recording
was shot. Command+Shift+3 will take a screen shot of the video, and then the same action
can be repeated at the 1 hour mark. The screenshots should then be cropped to fit the
hemocytometer and saved with the name of the petri dish, quadrant it was taken from, and
the times the screen shots were taken. (t=0 or t=1.) This should be repeated with the 9
remaining petri dishes.

It will take more than 10 hours to complete all 10 petri dishes of E. Coli, but it will
grant the best possible prediction for when the best time to infect the cells will be.
C A L C U L AT I N G T H E R AT E O F G R O W T H

(This is where a bit of beginner’s differential and integral calculus will come in handy. It’s okay if you
don’t know a lick of calculus; I already made the equation.)

This part of the procedure will allow the user to optimize the bacterial yield in the
cultures by using both science and basic differential/integral calculus techniques. The
following formula can be used to calculate how many cells there are in a single petri dish at
a particular time in hours;

# of cells = (cells per hour) * (hours)2 + (Initial amount of cells)*(hours)

2

To find the number of cells per hour, the following method should be used to establish
what is known as the slope of a secant line;

Cells per hour = (# of cells at T=1 hour) - (# of cells at T=0 hours)

This is not the most accurate way of estimating, but it will get a close enough estimate of
the number of cells for later.
INFECTING THE COLONIES

The goal is to have anywhere from 1 million cells per petri dish to 5 million cells per petri
dish before continuing to the next steps. The next steps will be to follow the instructions
provided by the two kits that will give the instructions for how to properly insert the
plasmid recombinant into the colonies, as well as how to then extract those proteins from
the colonies, which will be done two weeks apart, respectively. The materials needed for the
infection of the E. Coli colonies include;

• Custom DNA sequence from Genscript™ (The nucleotide sequence should be

TACGTCGCTATC which should translate to AUGCAGCGAUAG in RNA, and finally
Methionine-Glutamine-Arginine in amino acids.)

• Plasmid recombination kit

Instructions provided by the plasmid recombination kit from Bio-Rad (CAT#7326100)

will be followed since they apply to the materials supplied by the kit.

Only 9 petri dishes will be infected; the other petri dish is the control. 2 weeks should
pass before continuing to the next step.

EXTRACTING THE PROTEINS

The next step after the E. Coli have been infected and incubating for 2 more weeks
involves extraction of the proteins. The materials needed for this include;

• Centrifuge

• E. Coli colonies

• Pipettes

• Centrifuge tubes

• Protein extraction kit from Bio-Rad

 The instructions should be followed from the kit for the extraction process.
Centrifugation is required for the extraction and separation of the proteins. The kit is 201
USD but it will extract the proteins that are needed to perform the final step of this large
experiment.

TA G G I N G T H E A M I N O A C I D S

Tagging is a crucial step to ensure that the proper amino acids are present in the sample.
This will be done with the following materials;

• Ninhydrin

• Lab-Aids™ Thin-layer chromatography kit (219 USD)

Once again, the instructions provided by the kit will be followed. The Ninhydrin, however,
is used to mark the arginine amino acids in a sample, and will be done so by using the
instructions that come with the thin-layer chromatography kit.

ZINC-DIPEPTIDE REACTION

This is the part of the procedure that is going to determine as to whether or not there is a
chance for botox to have a binding affinity for the plain dipeptides in the suspension. The
following materials are needed;

• Elemental zinc powder (Ten 100 mg batches)

• Extracted peptide samples

• Water

• 2 100 mL beakers

• 1 mL pipette
 The first step will involve creating the suspension of zinc and extracted peptides. In
one 100 mL beaker, 19 mL of room temperature water will be added, and 1mL of the
extracted peptides should be added to the water. In the other beaker, 100 mg of elemental
zinc powder should be added to 1 mL of room temperature water. (The best way to do this
is to use water bottles and let them sit in the room all day before performing the
experiment.)

Once the two separate solutions are made, the polypeptide solution should be added to
the elemental zinc solution. Observations should be made and written down.

Once the visible reaction seems to have stopped, the beakers should be emptied, rinsed
thoroughly, and the procedure should be repeated again with varying concentrations. Let the
following table be an example;

Control 1x

19:1 (mL of water to mL of peptides.) 1x

18:2 1x

17:3 1x

16:4 1x

(And so on).

If this is successful, then the next step will be to return to the computer to begin mapping
out which amino acids should be used to assemble the protein cage, which is the next phase
of the procedure.
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A in Complex Biological Matrices. Retrieved March, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323579/

[2] Baldwin, M.R., Bradshaw, M., Johnson, E.A., & Barbier, J.T. (2004, September.). The C-Terminus of Botulinum
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[3] Chung, W., Sena, M., Merzlyak, A., & Lee, S. (2011). Phages as Tools for Functional Nanomaterials Development.
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[8] Lindsay, H. (2017, April 24). How to Make an Incubator to Grow Bacteria. Retrieved June 8, 2018, from https://
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[9] Parker, M. (2018, March 13). How to Grow E. Coli in a Petri Dish. Retrieved May 29, 2018, from https://sciencing.com/
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