1. Cyanobacterial Oscillator
The little oscillator that could...
Hetmann Hsieh
Jeff Lau
Dave Ramos
Zhipeng Sun
Harvard iGEM 2006

2. 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
Desirable properties:
Stable over time
Temperature compensated
Period can be adjusted via mutations in Kai genes (reference?) P P P P P P
Tomita et al, 2005

3. 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.

4. Experimental Overview
We measured the phosphorylation state of KaiC using Western blots
Problem: synchronization
Two-stage experimental design:
Interaction (unsynchronized)
Oscillation (synchronized)
Problem: can’t measure phosphorylation state of KaiC in an individual cell over time. Synchronize with pulses.
Interaction: Transform and grow E. coli cultures with different combinations of Kai genes. Verify expression of Kai genes in E. coli, look for unsynchronized interactions between Kai genes.
Oscillation: take time-series of synchronized E. coli cultures, look for oscillation

5. 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
KaiA + KaiC
high
KaiA + KaiB + KaiC
moderate

6. Results
We’ve made the following constructs:

7. Results Western blot results (interaction experiment)
Anti-KaiC antibodies

8. 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

9. 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 MIT and Eric WHOI for cyanobacteria strains