From cyanobacteria Photosynthetic Circadian rhythm Evolved over billions

From cyanobacteria. . . • Photosynthetic • Circadian rhythm • Evolved over billions of years . . . to E. coli • Model organism for synthetic biology • Bio. Brick registry Cyanobacterial Oscillator Harvard i. GEM 2006

Applications of a Bio-oscillator 12 PM A +E->F A +B->C 6 PM 6 AM • Clock • Nightlight • Timed drug delivery • Pharmaceutical processes • Bio-circuitry • Investigate natural systems 6 PM output D +C->E D +F->P 12 AM http: //www. cellzome. com/img/p athways. jpg Cyanobacterial Oscillator Harvard i. GEM 2006 Time

Cyanobacteria Bio-oscillator The Repressilator Lac λc. I Tet Fluorescence Elowitz et al. 2000 ` 10 h Cyanobacterial Oscillator Harvard i. GEM 2006 Time 24 h

The Kai Clock in Cyanobacteria P P P Phosphorylation of Kai. C • Kai. C autophosphorylates and dephosphorylates • Kai. A promotes phosphorylation • Kai. B inhibits Kai. A • Transcription-translation independent • Period: 14 -60 h b Time Cyanobacterial Oscillator Harvard i. GEM 2006

Achievements Goal: reconstitute the cyanobacteria Kai oscillator in E. coli 1. Created Kai. A, Kai. B, and Kai. C Bio. Bricks. 2. Combined the above with registry parts to form functional Bio. Bricks. 3. Expressed Kai proteins in E. coli and verified interaction. Cyanobacterial Oscillator Harvard i. GEM 2006

Results: Constructs Created We’ve made the following constructs: Kai genes Lac promoter + Kai genes Kai. A + Kai. C and Kai. B + Kai. C (with promoters) Cyanobacterial Oscillator Harvard i. GEM 2006

Results: Proteins Interact in E. coli Western blot image • Constructs transformed in E. coli • Cultures sampled at OD 0. 55 • Western blot washed with anti-Kai. C antibodies Cyanobacterial Oscillator Harvard i. GEM 2006

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

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

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

Results: Proteins Interact in E. coli Western blot image Conclusion: • Kai. A and Kai. C are expressed and interacting • Kai. B not verified, but results consistent with predictions Cyanobacterial Oscillator Harvard i. GEM 2006

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

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 • • Thanks for listening Harvard i. GEM 2006

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 Wiegele @ MIT and Eric Webb @ WHOI for cyanobacteria • Mark Howarth @ MIT and Filiz Aslan @ BU for streptavidin clones Word up Harvard i. GEM 2006