Download Constant KaiA, pulse KaiB and KaiC

Cyanobacterial Oscillator
The little oscillator that could...
Hetmann Hsieh
Jeff Lau
Dave Ramos
Zhipeng Sun
Harvard iGEM 2006
Cyanobacterial Oscillator
Harvard iGEM 2006
The Kai Clock in Cyanobacteria
Three proteins-- KaiA, KaiB, and KaiC– interact to produce oscillation in the
phosphorylation state of KaiC.
• Reconstituted in vitro with Kai genes
(Nakajima et al., 2005)
• Oscillation does not require
transcriptional regulation
P
• Desirable properties:
• Stable over time
P
• Temperature compensated
• Period can be adjusted via
P
mutations in Kai genes (reference?)
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P
P
Tomita et al, 2005
Cyanobacterial Oscillator
Harvard iGEM 2006
Project Goals
Reconstitute the cyanobacteria Kai oscillator in E. coli
1. Create KaiA, KaiB, and KaiC BioBricks.
2. Combine the above with promoters, etc. to form functional
BioBricks.
3. Express Kai proteins in E. coli and verify interaction.
4. Verify oscillation in E. coli.
Cyanobacterial Oscillator
Harvard iGEM 2006
Experimental Overview
• We measured the phosphorylation state of
KaiC using Western blots
– Problem: synchronization
– Solution: pulsed expression of Kai genes
• Two-stage experimental design:
– Interaction (unsynchronized)
– Oscillation (synchronized)
Cyanobacterial Oscillator
Harvard iGEM 2006
Interaction Experiments
According to Tomita et al.’s model of the Kai clock:
•KaiA promotes KaiC’s autophosphorylation
•KaiB inhibits KaiA
Experiments with constant (unsynchronized) expression of Kai genes:
Kai gene combination
Expected phosphorylation of
KaiC
KaiC
low
KaiB + KaiC
low
KaiA + KaiC
high
KaiA + KaiB + KaiC
moderate
Cyanobacterial Oscillator
Harvard iGEM 2006
Results
We’ve made the following constructs:
Cyanobacterial Oscillator
Harvard iGEM 2006
Results
Western blot results (interaction experiment)
Anti-KaiC antibodies
Cyanobacterial Oscillator
Harvard iGEM 2006
Future: Oscillation Experiments
• Induce expression of Kai genes
simultaneously for all cells in a culture
• Take samples at regular intervals
Pulse KaiA, KaiB, and KaiC expression
Expect: weakening oscillation
Time
Constant KaiA, pulse KaiB and KaiC
Expect: oscillation weakens more slowly than
above; period should not change
Cyanobacterial Oscillator
Harvard iGEM 2006
Acknowledgements
• Our TFs Chris Doucette, Shawn Douglas, and Nick
Stroustrup for their valuable help in lab
• Profs. Alain Viel, George Church, Pam Silver,
William Shih, Radhika Nagpal, and Jagesh Shah
• Prof. Susan Golden at TAMU for providing Western
blot antibodies and advice
• Peter Wiegele @ MIT and Eric Webb @ WHOI for
cyanobacteria strains
Cyanobacterial Oscillator
Harvard iGEM 2006