Harvard iGEM 2006 Cyanobacterial Oscillator From cyanobacteria......to E. coli Photosynthetic Circadian rhythm Evolved over billions of years Model organism for synthetic biology BioBrick registry

Harvard iGEM 2006 Cyanobacterial Oscillator Time Applications of a Bio-oscillator hu -Clock -Nightlight -Timed drug delivery -Bio-circuitry -Scientific research

Harvard iGEM 2006 Cyanobacterial Oscillator The Repressilator Our Bio-oscillator Lac Λ- cI Tet Time Fluorescence Elowitz et al. 2000

Harvard iGEM 2006 Cyanobacterial Oscillator PP P P P P The Kai Clock in Cyanobacteria KaiC autophosphorylates and dephosphorylates KaiA promotes phosphorylation KaiB inhibits KaiA Time Phosphorylation of KaiC

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.

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Construct Creation We’ve made the following constructs: Kai genes Lac promoter + Kai genes KaiA + KaiC and KaiB + KaiC (with promoters)

Harvard iGEM 2006 Cyanobacterial Oscillator PP P P P P Results: Protein Interaction

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Protein Interaction Constructs were transformed in E. coli Cultures were grown overnight Western blot was washed with anti-KaiC antibodies (courtesy of Susan Golden) The gel was imaged, producing the above results containing phosphorylated and unphosphorylated KaiC Western blot Image Anti-KaiC antibodies

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Protein Interaction Western blot results Anti-KaiC antibodies PP P P P P

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Protein Interaction Western blot results (interaction experiment) Anti-KaiC antibodies PP P P P P

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Protein Interaction Western blot results (interaction experiment) Anti-KaiC antibodies PP P P P P

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Protein Interaction Western blot results (interaction experiment) Anti-KaiC antibodies PP P P P P

Harvard iGEM 2006 Cyanobacterial Oscillator Future work Verify oscillation in E. coli. Synchronization problem Within cell Between cells Solution: Pulsed expression of Kai genes

Harvard iGEM 2006 Cyanobacterial Oscillator Future work Verify oscillation in E. coli. Synchronization problem Within cell Between cells Solution: Pulsed expression of Kai genes

Harvard iGEM 2006 Cyanobacterial Oscillator Acknowledgements Our teaching fellows 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 Texas A&M for providing anti- KaiC antibodies and advice Peter MIT and Eric WHOI for cyanobacteria strains