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From cyanobacteria... • Photosynthetic • Circadian rhythm • Evolved over billions of years ...to E. coli • Model organism for synthetic biology • BioBrick registry Word up Harvard iGEM 2006 Applications of a Bio-oscillator 12PM 12PM 6PM 6AM 12AM 12AM Time Word up Harvard iGEM 2006 6PM -Clock -Nightlight -Timed drug delivery -Bio-circuitry -Scientific research Cyanobacterial Bio-oscillator The Repressilator Lac λcI Tet Elowitz et al. 2000 Fluorescence ` Time Word up Harvard iGEM 2006 10h The Kai Clock in Cyanobacteria P P P P P P Phosphorylation of KaiC •KaiC autophosphorylates and dephosphorylates •KaiA promotes phosphorylation •KaiB inhibits KaiA •Transcription-translation independent •Period: 14-60 h b Time Word up Harvard iGEM 2006 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. Word up Harvard iGEM 2006 Results: Constructs Created We’ve made the following constructs: Kai genes Lac promoter + Kai genes KaiA + KaiC and KaiB + KaiC (with promoters) Word up Harvard iGEM 2006 Results: Proteins Interact in E. coli Western blot Image • Constructs were transformed in E. coli • Cultures were grown for 16 hours • Western blot was washed with anti-KaiC antibodies Word up Harvard iGEM 2006 Results: Proteins Interact in E. coli Western blot Image P P P P P Word up Harvard iGEM 2006 P Results: Proteins Interact in E. coli Western blot Image P Word up Harvard iGEM 2006 Results: Proteins Interact in E. coli Western blot Image P P P P P Word up Harvard iGEM 2006 P Results: Proteins Interact in E. coli Western blot Image Anti-KaiC antibodies P P P P P Word up Harvard iGEM 2006 P Results: Proteins Interact in E. coli Western blot Image Anti-KaiC antibodies Conclusion: • KaiA and KaiC are expressed and interacting • KaiB not verified, but results consistent with predictions Word up Harvard iGEM 2006 Further challenges Phosphorylated KaiC (constant production) Synchronization problem • Between cells • Within cell Percentage of phosphorylated KaiC Verify oscillation in E. coli. 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 25 49 73 97 121 145 169 193 217 Time (h) Time Word up Harvard iGEM 2006 Output from a computer model simulating KaiC oscillation in a single cell Conclusion Word up Harvard iGEM 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 iGEM 2006
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