Cyanobacterial Oscillator

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

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.

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

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

Results We’ve made the following constructs:

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

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

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