Transcript 1-2 wks - OpenWetWare

and could…
and could…
The little oscillator that could.
and could…
and could…
http://www.worldofbubble.com/thomas_tank_engine/thomas.html
• Some strains of cyanobacteria oscillate on a circadian cycle. This
cycle is driven by the interaction of three proteins, KaiABC, which
are sufficient to produce oscillation in vitro without transcription
regulation (Nakajima et al., 2005).
• Cyanobacteria oscillation is robust and temperature-independent
(within living tolerances).
• The oscillation period can be adjusted from 14-60hours by point
mutations of KaiC (Kondo et al., 2000).
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Transcriptional repression system
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T~200min
Is not stable over time
Advantages of Cyanobacteria oscillator
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Plasmid to right, GFP reporter
‘Lite’ means destruction tag
Stable over time
Potentially more robust due to
evolutionary development
Post translational mechanism means less
energy?
Problem with implementation in later
generations
According to Elowitz:
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“However, the reliable performance of
[cyanobacteria] circadian oscillators can
be contrasted with the noisy, variable
behavior of the repressilator…It would be
interesting to see whether one could
build an artificial analogue of the
circadian clock.”
Deliverable: Bacterial Nightlight in E. coli
Fallback: Bacterial Nightlight in Cyanobacteria
Intermediate Goals:
• Use Kai sequence to create a functional
oscillator Biobrick.
• Use a luciferase gene reporter to
measure Kai activity (e.g. GFP).
• Use oscillator with luciferase to
construct a nightlight.
http://www.footvolley.net/images/ronaldo%20world%20cup%20goal.jpg
Obtain an appropriate strand of cyanobacteria (1-2 wks)
1.
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Extract the KaiABC genes from cyanobacteria and biobrick them (12 wks)
2.
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Design of primers can be done beforehand
Design a feasible E. coli sequence for KaiABC, and synthesize it (can
be done in parallel with step 1) (1-2 wks)
3.
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4.
Contact MIT iGem team for leads
Synechococcus PCC7942 or WH8102
Research the modifications we will need to make to the cyanobacteria
genes to make them compatible with E. Coli; if they’re small, we won’t
need to synthesize the whole sequence.
Instead of synthesizing entire 3kb sequence, break into smaller
sequences to be synthesized separately to save on cost, and recombine
by PCR.
Insert both sequences (synthesized and BioBrick’d from
cyanobacteria) into E. Coli and test (5+ wks)
http://www.compendia.co.uk/acatalog/risk.jpg
 There is a known codon bias problem with 2 amino acids
 Possible resolution to codon bias: we can synthetically
modify the codons for the 2 amino acids to be
compatible in e. coli
 Other environmental factors in E. coli may hinder the
oscillator
 More proteins may be involved than KaiABC
 But KaiABC have been shown to work in vitro
 Problems with synthesis of KaiABC
 Not obtaining the cyanobacteria from various sources
 This can be resolved by using alternative methods
of synthesis
 Testing
 Test three plasmids attached to KaiA, KaiB, and KaiC
 Loss of time, effort, and resources
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Resolved by implementing intermediate goals such
as a "nightlight" in cyanobacteria, not E. coli
Problem:
Not obvious how to wire clock output to other cell activities (like
transcription) in E. coli without the complex and partially nebulous
circadian elements in cyanobacteria.
Possible Solutions:
Directly measure the amount of KaiC phosphorylation using
antibody staining. But this doesn’t help us make the cell do any
useful work.
Robustness:
The repressilator destabilizes over time, but our oscillator will retain
its period and amplitude after long periods of time.
Variability:
Can experimentally vary the period of oscillation from 14h to 60h
(Kondo et. al 2000) with KaiC point mutations.
Useful Applications:
Can implement a clock or timer in gene circuits analogous to similar
parts in silico, and trigger events at certain times.
iGem Performance:
A robust Biobricked oscillator, and its application in our system, will
impress iGem judges.
Team effort:
Creating a working oscillator will require each of us to contribute our
respective strengths: C.S. for modeling, Biochemistry for
understanding and implementing the circuitry.
Fun Factor:
Strong.