Transcript The circadian clock of cyanobacteria.

and could…
and could…
The little oscillator that could.
and could…
and could…
http://www.worldofbubble.com/thomas_tank_engine/thomas.html
• Cyanobacteria display a circadian rhythm. In some strains, this
rhythm has been shown to be 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 of the repressillator
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-day)
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Extract the KaiABC genes from cyanobacteria and biobrick them (12 wks)
Design a feasible E. coli sequence for KaiABC, and synthesize it
(can be done in parallel with steps 1 & 2) (1-2 wks)
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Synechococcus PCC7942 or WH8102
Peter Weigle has it… or do you have a copy of it, Prof. Church?
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)
 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
 Environmental factors within E. coli may hinder the
oscillator
 More proteins may be involved than KaiABC
 But KaiABC have been shown to work in vitro
 Costs of KaiABC synthesis.
http://www.compendia.co.uk/acatalog/risk.jpg
 Test three plasmids attached to KaiA, KaiB, and
KaiC.
 Loss of time, effort, and resources due to
implementing system in new environment.
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Resolved by implementing fallback 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.
Kondo et al
2004
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.
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Kucho K, Okamoto K, Tsuchiya Y, Nomura S, Nango M, Kanehisa M, and Ishiura M. Global
analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol
2005 Mar; 187(6) 2190-9. doi:10.1128/JB.187.6.2190-2199.2005 pmid:15743968.
Takigawa-Imamura H and Mochizuki A. Transcriptional autoregulation by phosphorylated and
non-phosphorylated KaiC in cyanobacterial circadian rhythms. J Theor Biol 2005 Dec 29.
doi:10.1016/j.jtbi.2005.11.013 pmid:16387328.
Kutsuna S, Nakahira Y, Katayama M, Ishiura M, and Kondo T. Transcriptional regulation of the
circadian clock operon kaiBC by upstream regions in cyanobacteria. Mol Microbiol 2005 Sep; 57(5)
1474-84. doi:10.1111/j.1365-2958.2005.04781.x pmid:16102014.
Wang J. Recent cyanobacterial Kai protein structures suggest a rotary clock. Structure 2005 May;
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Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, and Kondo T.
Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 2005
Apr 15; 308(5720) 414-5. doi:10.1126/science.1108451 pmid:15831759. PubMed HubMed [2]
Imai K, Nishiwaki T, Kondo T, and Iwasaki H. Circadian rhythms in the synthesis and degradation
of a master clock protein KaiC in cyanobacteria. J Biol Chem 2004 Aug 27; 279(35) 36534-9.
doi:10.1074/jbc.M405861200 pmid:15229218. PubMed HubMed [3]
Naef F. Circadian clocks go in vitro: purely post-translational oscillators in cyanobacteria. Mol Syst
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