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

2. Harvard iGEM 2006 Motivation Cyanobacterial Oscillator 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. Harvard iGEM 2006 The Kai clock in cyanobacteria Cyanobacterial Oscillator Three proteins-- KaiA, KaiB, and KaiC– interact to produce oscillation in the phosphorylation state of KaiC. Tomita et al, 2005 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)

4. Harvard iGEM 2006 Project Goals Cyanobacterial Oscillator Reconstitute the cyanobacteria KaiABC oscillator in E. coli 1.Create KaiA, KaiB, and KaiC Biobricks. 2.Ligate KaiABC BioBricks with promoters, etc. to form functional constructs. 3.Transform E. coli with Kai constructs to reconstitute KaiC phosphorylation cycle (no reporter attached). 4.Distant: Implement intermediate transcription factors so the Kai clock can do useful work in E. coli.

5. Harvard iGEM 2006 Obtaining KaiABC: Extraction Cyanobacterial Oscillator 5’ 3’ kaiBC kaiA EcoRIXbaI SpeI 3 EcoRIPstI A 3.1kb template containing KaiA, B, and C from cyanobacteria AdvantageDisadvantage Can be made into a biobrick compatible with cyanobacteria as well as basis for E. coli version Need three point mutations Need to split into KaiA, B, and C for insertion into e. coli Result: Had problems with cloning; postponed until later

6. Harvard iGEM 2006 Obtaining KaiABC: Synthesis Cyanobacterial Oscillator Three separate constructs for each of KaiA, KaiB, and KaiC with biobrick ends Synthesis from Geneart GMBH AdvantageDisadvantage Relatively fast (14 business days) Cannot make a cyanobacteria- compatible biobrick $$$ Result: Went with synthetic genes

7. Harvard iGEM 2006 Experiments: Interaction of KaiABC Cyanobacterial Oscillator R0010B0034 R0010: lac promoter B0034: strong RBS KaiC never phosphorylates KaiA phosphorylates KaiC KaiB dephosphorylates KaiC Time

8. Harvard iGEM 2006 Experiments: Oscillation of KaiABC Cyanobacterial Oscillator Pulse KaiA once and pulse KaiB + KaiC once Oscillation should weaken as KaiC degrades R0010B0034 B0010 Constant KaiA, pulse KaiB + KaiC once If KaiA phosphorylation and KaiC degradation at equal rates, should oscillate Time

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

10. Harvard iGEM 2006 Acknowledgements Cyanobacterial Oscillator 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