Hetmann Hsieh Jeffrey Lau David Ramos Zhipeng Sun

Outline: Introduction Motivation Kai Protein Interactions Project Goals Obtaining the Kai Proteins Biobricking the Kai Proteins Stage I: Expression and Interaction of Kai Proteins in E. coli Stage II: Synchronization of Kai Proteins in E. coli Stage III: Future Possibilities Conclusion

The repressilator: Transcriptional repression system Plasmid to right, GFP reporter ‘Lite’ means destruction tag T~200min Is not stable over time Problem with implementation in later generations of the repressillator Why a cyanobacterial oscillator instead? Rhythm has been shown to be driven by the interaction of three proteins, KaiABC, which are sufficient to produce oscillation in vitro without transcription regulation (Nakajima et al., 2005). Cyanobacteria oscillation is robust, stable over time, and temperature-independent (within living tolerances). The oscillation period can be adjusted from 14-60hours by point mutations of KaiC (Kondo et al., 2000). Post translational mechanism means less energy required. (keep?) Elowitz et al. 2000

Reconstitute the cyanobacteria KaiABC oscillator in E. coli 1.Create KaiA, KaiB, and KaiC Biobricks. 2.Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle (no reporter attached). 3.Distant: Transform E. coli with Kai Briobricks to reconstitute KaiC phosphorylation cycle with Biobricked reporter.

Extracting from PCC7942 cyanobacteria:

DNA synthesis: Synthesized KaiA, KaiB, and KaiC separately, with biobrick prefixes and suffixes and extra stop codons. Also included two point mutations on KaiC to remove the PstI and EcoRI sites to ensure biobricks compatibility. Ordered from Geneart and received correct sequences in 18 days.