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From cyanobacteria... • Photosynthetic • Circadian rhythm • Evolved over billions of years ...to E. coli • Model organism for synthetic biology • BioBrick registry Cyanobacterial Oscillator Harvard iGEM 2006 Applications of a Bio-oscillator 12PM 12PM 6PM 6AM 12AM 12AM Time Cyanobacterial Oscillator 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 Cyanobacterial Oscillator 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 Cyanobacterial Oscillator 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. Cyanobacterial Oscillator Harvard iGEM 2006 Results: Construct Creation We’ve made the following constructs: Kai genes Lac promoter + Kai genes KaiA + KaiC and KaiB + KaiC (with promoters) Cyanobacterial Oscillator Harvard iGEM 2006 Results: Protein Interaction Western blot Image • Constructs were transformed in E. coli • Cultures were grown for 16 hours • Western blot was washed with anti-KaiC antibodies (courtesy of Professor Susan Golden at Texas A&M) Cyanobacterial Oscillator Harvard iGEM 2006 Results: Protein Interaction Western blot Image P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot Image P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot Image P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot Image Anti-KaiC antibodies P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot Image Anti-KaiC antibodies Conclusion: • KaiA and KaiC expressed and interacting • KaiB not verified, but consistent with prediction Cyanobacterial Oscillator 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 Cyanobacterial Oscillator Harvard iGEM 2006 Output from a computer model simulating KaiC oscillation in a single cell Conclusion Thanks for listening Harvard iGEM 2006 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 antiKaiC antibodies and advice • Peter Wiegele @ MIT and Eric Webb @ WHOI for cyanobacteria strains Thanks for listening Harvard iGEM 2006
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