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 Clock Nightlight Timed drug delivery Pharmaceutical processes Bio-circuitry Investigate natural systems 12PM 6PM6AM6PM 12AM output pathways.jpg

Harvard iGEM 2006 Cyanobacterial Oscillator The Repressilator Cyanobacteria Bio-oscillator Lac λ - cI Tet Elowitz et al ` Time Fluorescence 10h24h

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 Transcription-translation independent Period: h Time Phosphorylation of KaiC b

Harvard iGEM 2006 Cyanobacterial Oscillator Achievements Goal: reconstitute the cyanobacteria Kai oscillator in E. coli 1.Created KaiA, KaiB, and KaiC BioBricks. 2.Combined the above with registry parts to form functional BioBricks. 3.Expressed Kai proteins in E. coli and verified interaction.

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

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Proteins Interact in E. coli Constructs transformed in E. coli Cultures sampled at OD 0.55 Western blot washed with anti-KaiC antibodies Western blot image

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Proteins Interact in E. coli Western blot image P P P P P P P PP

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Proteins Interact in E. coli Western blot image P P P P P P P PP

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Proteins Interact in E. coli Western blot image P P P P P P P PP

Harvard iGEM 2006 Cyanobacterial Oscillator Results: Proteins Interact in E. coli Western blot image Conclusion: KaiA and KaiC are expressed and interacting KaiB not verified, but results consistent with predictions

Harvard iGEM 2006 Cyanobacterial Oscillator PP P P P P Further challenges Verify oscillation in E. coli. Synchronization problem Between cells Within cell Time Solution: pulsed expression

Harvard iGEM 2006 Thanks for listening Conclusion be sure not to say not the only oscillating bacteria “do not say more scienfitic” – say investiage natural systems More stuff on the scientific/pharmacutical Say “We interpret these bands as showing...” not that “they are phosphorylated bands” Future work: sentence about what the experiment is (syncronize cultures by pulsing inducible promoters) say stable over time mention that we added parts to the registry

Harvard iGEM 2006 Word up Acknowledgements Our teaching fellows: Chris Doucette, Shawn Douglas, and Nick Stroustrup Our advisors: Profs. Alain Viel, George Church, Pam Silver, William Shih, Radhika Nagpal, and Jagesh Shah Prof. Susan Golden at Texas A&M for advice and antibodies Peter MIT and Eric WHOI for cyanobacteria Mark MIT and Filiz BU for streptavidin clones