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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 -Clock -Nightlight hu -Timed drug delivery -Bio-circuitry -Scientific research Time Cyanobacterial Oscillator Harvard iGEM 2006 The Repressilator Cyanobacterial Oscillator Lac ΛcI Tet Fluorescence Elowitz et al. 2000 Time Cyanobacterial Oscillator Harvard iGEM 2006 The Kai Clock in Cyanobacteria P P P P P P Phosphorylation of KaiC •KaiC autophosphorylates and dephosphorylates •KaiA promotes phosphorylation •KaiB inhibits KaiA •Necessary and sufficient •Transcription independent 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 P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot Image Anti-KaiC antibodies • Constructs were transformed in E. coli • Cultures were grown overnight • Western blot was washed with anti-KaiC antibodies (courtesy of Susan Golden) • The gel was imaged, producing the above results containing phosphorylated and unphosphorylated KaiC Cyanobacterial Oscillator Harvard iGEM 2006 Results: Protein Interaction Western blot results Anti-KaiC antibodies P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot results (interaction experiment) Anti-KaiC antibodies P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot results (interaction experiment) Anti-KaiC antibodies P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Results: Protein Interaction Western blot results (interaction experiment) Anti-KaiC antibodies P P P P P Cyanobacterial Oscillator Harvard iGEM 2006 P Future work Verify oscillation in E. coli. Solution: Pulsed expression of Kai genes Percentage of phosphorylated KaiC Synchronization problem • Within cell • Between cells Phosphorylated KaiC (constant production) 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 Time (h) Cyanobacterial Oscillator Harvard iGEM 2006 145 169 193 217 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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