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From cyanobacteria...

From cyanobacteria...

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From cyanobacteria...

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  1. From cyanobacteria... • Photosynthetic • Circadian rhythm • Evolved over billions of years ...to E. coli • Model organism for synthetic biology • BioBrick registry

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

  3. The Repressilator Cyanobacteria Bio-oscillator Lac λ-cI Tet Elowitz et al. 2000 ` Fluorescence Time 10h 24h

  4. The Kai Clock in Cyanobacteria KaiCautophosphorylates and dephosphorylates KaiA promotes phosphorylation KaiB inhibits KaiA Transcription-translation independent Period: 14-60 h A B C P P P P P P b Phosphorylation of KaiC output Time

  5. Achievements Goal: reconstitute the cyanobacteria Kai oscillator in E. coli Created KaiA, KaiB, and KaiC BioBricks. Combined the above with registry parts to form functional BioBricks. Expressed Kai proteins in E. coli and verified interaction.

  6. Results: Constructs Created We’ve made the following constructs: Kai genes Lac promoter + Kai genes KaiA + KaiC and KaiB + KaiC (with promoters)

  7. 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-KaiC antibodies

  8. Results: Proteins Interact in E. coli Western blot image A A P P P P P P P P P C B C C

  9. Results: Proteins Interact in E. coli Western blot image A A P P P P P P P P P C B C C

  10. Results: Proteins Interact in E. coli Western blot image A A P P P P P P P P P C B C C

  11. 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

  12. Further challenges A B A A A B B B C C C C A Time B C Verify oscillation in E. coli. Synchronization problem • Between cells • Within cell Solution: pulsed expression P P P P P P

  13. 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

  14. 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