1. Harvard iGEM 2006 Cyanobacterial Oscillator Hetmann Hsieh Jeff Lau Dave Ramos Zhipeng Sun Harvard iGEM 2006 Creating a robust oscillator BioBrick

2. Harvard iGEM 2006 Cyanobacterial Oscillator Motivation Repressilator (Elowitz and Leibler, 2000) – Synthetic oscillator – Unstable over time

3. Harvard iGEM 2006 Cyanobacterial Oscillator The Kai Clock in Cyanobacteria Three proteins-- KaiA, KaiB, and KaiC– interact to produce oscillation in the phosphorylation state of KaiC. Tomita et al, 2005 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?) PPPP PPPP PPPP

4. Harvard iGEM 2006 Cyanobacterial Oscillator 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. 5.(Future) Implement intermediate transcription factors so the Kai clock can do useful work in E. coli.

5. Harvard iGEM 2006 Cyanobacterial Oscillator Experimental Overview We measured the phosphorylation state of KaiC using Western blots – Problem: synchronization Two-stage experimental design: – Interaction (unsynchronized) – Oscillation (synchronized)

6. Harvard iGEM 2006 Cyanobacterial Oscillator 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 combinationExpected phosphorylation of KaiC KaiClow KaiB + KaiClow KaiA + KaiChigh KaiA + KaiB + KaiCmoderate

7. Harvard iGEM 2006 Cyanobacterial Oscillator Oscillation Experiments Time 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 Constant KaiA, pulse KaiB and KaiC Expect: oscillation weakens more slowly than above; period should not change

8. Harvard iGEM 2006 Cyanobacterial Oscillator Results We’ve made the following constructs: Western blot results (interaction experiment) Anti-KaiC antibodies

9. Harvard iGEM 2006 Cyanobacterial Oscillator Future Work More constructs to build: Oscillation experiments

10. Harvard iGEM 2006 Cyanobacterial Oscillator 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