Preparation of

competent Escherichia
coli cells
By: Dr. Shahjahan
Associate Professor,

FLS&I, BUITEMS

E. coli competence project

Background
Generally > 35 % post-harvest losses
of horticultural commodities are
reported annually in Pakistan
2 basic strategies of management
can be adapted

E. coli competence project

Physiological Strategy
Management of fruits at physiological level is
widely studied
Storage life can be prolonged by
Harvesting at proper maturity
Control of post harvest diseases
Regulation of atmosphere, chemical
treatments, irradiation, refrigeration, etc.
Control of modified atmosphere
Several other treatments like waxing,
grading, packaging, etc.
E. coli competence project

Main goals of storage

1. Slow the biological activity of fruits
and vegetables without chilling
injury
2. Slow the growth of microorganisms
3. Reduce the transpiration losses

E. coli competence project

Critical physiological factors

related to ripening
Relative Humidity (RH %)
Temperature (C, F)
Gaseous exchange like (O2, CO2,
C2H4, N2)
Controlled (CA) and Metabolic (MA)
systems
Controlled modified atmosphere
(Hypobaric storage/vacuum storage),
etc.
E. coli competence project

Metabolic effect of CA
storage
Retard respiration
Cause acid accumulation and
acetaldehyde formation
Increase sugars
Decrease alcohol soluble and protein
N
Cause Pectin changes
Inhibit chlorophyll degradation
E. coli competence project

Metabolic effect of high CO2

levels on fruits & vegetables
1.
2.
3.
4.
5.
6.

Ripening
Enzyme activity
Production of volatile
Metabolism of organic acids
Breakdown of pectic substances
Chlorophyll synthesis & fruit
degreening
E. coli competence project

Toxic effects of CA
High level of CO2 and low level of O2
may cause
1. Decay
2. Internal browning
3. Breakdown and accumulation of
some organic acids such as succinic
acid of toxic levels (0.001 M)

E. coli competence project

Molecular Strategy
Hydrolytic enzymes like pectinases,
polygalacturonases (PG),
polyphenoloxidases, peroxidases at
different levels of ethylene and respiration
rates express and behave differently
One of the paramount enzymes involved
in ripening, softening and aging of fruits is
PG, which can be masked by Gene
Silencing
E. coli competence project

Objectives
Develop cells working as vehicles to
carry RNA and DNA duplexes
Develop plasmid vectors for gene
expression and silencing analysis
Set up gene transformation and
delivery system at BUITEMS

E. coli competence project

Circular map of pUC18

E. coli competence project

RNA Interference (RNAi)

Andrew Fire and Craig C. Mellow have
been awarded the Noble prize in
Physiology and Medicine in 2006 for their
discovery of (RNAi)
They deciphered RNAi into Caenorhabditis
elegans nematode blocks specific genes
expression and that inhibition persists
well into the next generation
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RNAi Mechanism

E. coli competence project

RNAi Mechanism

GS Bio, incorporation Lab,

E. coli competence project

E. coli competence project

E. coli competence project

E. coli competence project

Current status of
research
E. coli has been developed for
harbouring pUC18
PG-PCR optimization after cDNA
preparation in progress
pUC18 has been mapped and
digested with restriction enzymes
pUC18 with PG-mRNA hybrid will be
designed
E. coli competence project

Thank you

E. coli competence project