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Cyanobacterial Oscillator

Cyanobacterial Oscillator. Harvard iGEM 2006. Cyanobacterial Oscillator. Creating a robust biological oscillating biobrick. Hetmann Hsieh Jeff Lau Dave Ramos Zhipeng Sun Harvard iGEM 2006. Cyanobacterial Oscillator. Harvard iGEM 2006. Motivation.

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Cyanobacterial Oscillator

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  1. Cyanobacterial Oscillator Harvard iGEM 2006 Cyanobacterial Oscillator Creating a robust biological oscillating biobrick. Hetmann Hsieh Jeff Lau Dave Ramos Zhipeng Sun Harvard iGEM 2006

  2. Cyanobacterial Oscillator Harvard iGEM 2006 Motivation • Repressilator (Elowitz and Leibler, 2000) • Synthetic oscillator • Unstable over time • Our cyanobacterial oscillator • Stable over long periods of time • Period flexible from 14-60h

  3. Cyanobacterial Oscillator Harvard iGEM 2006 The Kai clock in cyanobacteria Three proteins-- KaiA, KaiB, and KaiC– interact to produce oscillation in the phosphorylation state of KaiC. • Reconstituted in vitro with KaiABC proteins (Nakajima et al., 2005) • Oscillation does not require transcriptional regulation • Oscillation robust and temperature-resistant • Many processes are regulated by the Kai phosphorylation cycle in cyanobacteria (including transcription of KaiBC) Tomita et al, 2005

  4. Cyanobacterial Oscillator Harvard iGEM 2006 Project Goals • Reconstitute the cyanobacteria KaiABC oscillator in E. coli • Create KaiA, KaiB, and KaiC Biobricks. • Ligate KaiABC BioBricks with promoters, etc. to form functional constructs. • Transform E. coli with Kai constructs to reconstitute KaiC phosphorylation cycle (no reporter attached). • Distant: Implement intermediate transcription factors so the Kai clock can do useful work in E. coli.

  5. Cyanobacterial Oscillator Harvard iGEM 2006 Obtaining KaiABC: Extraction PstI EcoRI XbaI EcoRI PstI kaiBC kaiA 5’ 3’ 3’ 5’ SpeI 3

  6. Cyanobacterial Oscillator Harvard iGEM 2006 Obtaining KaiABC: Synthesis

  7. Cyanobacterial Oscillator Harvard iGEM 2006 Experiments: Interaction of KaiABC R0010 B0034 • R0010: lac promoter • B0034: strong RBS • KaiC never phosphorylates • KaiA phosphorylates KaiC • KaiB dephosphorylates KaiC Time

  8. Cyanobacterial Oscillator Harvard iGEM 2006 Experiments: Oscillation of KaiABC R0010 B0034 B0010 • Pulse KaiA once and pulse KaiB + KaiC once • Oscillation should weaken as KaiC degrades Time • Constant KaiA, pulse KaiB + KaiC once • If KaiA phosphorylation and KaiC degradation at equal rates, should oscillate

  9. Cyanobacterial Oscillator Harvard iGEM 2006 Results When we get the KaiC construct working this will be filled… :D

  10. Cyanobacterial Oscillator Harvard iGEM 2006 Acknowledgements • Our TF’s 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 TAMU for helping us with Western blot antibodies and general advice • Peter Wiegele @ MIT and Eric Webb @ WHOI for PCC7942 and PCC6803 strains

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