Cyanobacterial Oscillator

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Cyanobacterial Oscillator
The little oscillator that could...

• Hetmann Hsieh
• Jeff Lau
• Dave Ramos
• Zhipeng Sun
• Harvard iGEM 2006

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The Kai Clock in Cyanobacteria
Three proteins-- KaiA, KaiB, and KaiC interact
to produce oscillation in the phosphorylation
state of KaiC.
A

• Reconstituted in vitro with Kai genes (Nakajima
  et al. 2005)
• Oscillation does not require transcriptional
  regulation
• The cyanobacterial oscillator is
• Stable over time
• Temperature compensated
• Period can be adjusted via mutations in Kai
  genes (Kondo et al. 1994)

B
C
C
Tomita et al. 2005
TF?
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Project Goals

• Reconstitute the cyanobacteria Kai oscillator in
  E. coli
• Create KaiA, KaiB, and KaiC BioBricks.
• Combine the above with promoters, etc. to form
  functional BioBricks.
• Express Kai proteins in E. coli and verify
  interaction.
• Verify oscillation in E. coli.

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Experimental Overview

• We measured the phosphorylation state of KaiC
  using Western blots
• Synchronization problem
• Between cells
• Within cells
• Solution pulsed expression of Kai genes
• Two-stage experimental design
• Interaction (unsynchronized)
• Oscillation (synchronized)

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Interaction Experiments

• According to Tomita et al.s model of the Kai
  clock
• KaiA promotes KaiCs 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
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Results

• Weve made the following constructs

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Western Blot Results
with Anti-KaiC antibodies
1.5x
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Future Oscillation Experiments

• Induce expression of Kai genes simultaneously
  for all cells in a culture
• Take samples at regular intervals

KaiC phosphorylation
Pulse KaiA, KaiB, and KaiC expression Expect
weakening oscillation
Time
KaiC phosphorylation
Constant KaiA, pulse KaiB and KaiC Expect
oscillation weakens more slowly than above
period should not change
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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 _at_ MIT and Eric Webb _at_ WHOI for
  cyanobacteria strains