The little oscillator that could.and could Cyanobacteria display a circadian rhythm. In some strains, this 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 and temperature-independent (within living tolerances). The oscillation period can be adjusted from 14-60hours by point mutations of KaiC (Kondo et al., 2000). Transcriptional repression system Plasmid to right, GFP reporter Lite means destruction tag T~200min Is not stable over time Advantages of Cyanobacteria oscillator Stable over time Potentially more robust due to evolutionary development Post translational mechanism means less energy? Problem with implementation in later generations of the repressillator According to Elowitz: However, the reliable performance of [cyanobacteria] circadian oscillators can be contrasted with the noisy, variable behavior of the repressilatorIt would be interesting to see whether one could build an artificial analogue of the circadian clock. Intermediate Goals: Use Kai sequence to create a functional oscillator Biobrick. Use a luciferase gene reporter to measure Kai activity (e.g. GFP). Use oscillator with luciferase to construct a nightlight.Deliverable: Bacterial Nightlight in E. coli Fallback: Bacterial Nightlight in Cyanobacteria 1. Obtain an appropriate strand of cyanobacteria (1-day) Synechococcus PCC7942 or WH8102 Peter Weigle has it or do you have a copy of it, Prof. Church? 2. Extract the KaiABC genes from cyanobacteria and biobrick them (1- 2 wks) 3. Design a feasible E. coli sequence for KaiABC, and synthesize it (can be done in parallel with steps 1 & 2) (1-2 wks) Research the modifications we will need to make to the cyanobacteria genes to make them compatible with E. Coli; if theyre small, we wont need to synthesize the whole sequence. Instead of synthesizing entire 3kb sequence, break into smaller sequences to be synthesized separately to save on cost, and recombine by PCR. 4. Insert both sequences (synthesized and BioBrickd from cyanobacteria) into E. Coli and test (5+ wks) There is a known codon bias problem with 2 amino acids Possible resolution to codon bias: we can synthetically modify the codons for the 2 amino acids to be compatible in e. coli Environmental factors within E. coli may hinder the oscillator More proteins may be involved than KaiABC But KaiABC have been shown to work in vitro Costs of KaiABC synthesis. Test three plasmids attached to KaiA, KaiB, and KaiC. Loss of time, effort, and resources due to implementing system in new environment. Resolved by implementing fallback goals such as a "nightlight" in cyanobacteria, not E. coli Problem: Not obvious how to wire clock output to other cell activities (like transcription) in E. coli without the complex and partially nebulous circadian elements in cyanobacteria. Possible Solutions: Directly measure the amount of KaiC phosphorylation using antibody staining. But this doesnt help us make the cell do any useful work. Kondo et al 2004 Robustness:The repressilator destabilizes over time, but our oscillator will retain its period and amplitude after long periods of time. Variability:Can experimentally vary the period of oscillation from 14h to 60h (Kondo et. al 2000) with KaiC point mutations. Useful Applications:Can implement a clock or timer in gene circuits analogous to similar parts in silico, and trigger events at certain times. iGem Performance:A robust Biobricked oscillator, and its application in our system, will impress iGem judges. Team effort:Creating a working oscillator will require each of us to contribute our respective strengths: C.S. for modeling, Biochemistry for understanding and implementing the circuitry. Fun Factor:Strong. 1. Kondo T and Ishiura M. The circadian clock of cyanobacteria. Bioessays 2000 Jan; 22(1) doi: /(SICI) (200001)22:1 3.0.CO;2-A pmid: Kucho K, Okamoto K, Tsuchiya Y, Nomura S, Nango M, Kanehisa M, and Ishiura M. Global analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC J Bacteriol 2005 Mar; 187(6) doi: /JB pmid: Takigawa-Imamura H and Mochizuki A. Transcriptional autoregulation by phosphorylated and non-phosphorylated KaiC in cyanobacterial circadian rhythms. J Theor Biol 2005 Dec 29. doi: /j.jtbi pmid: Kutsuna S, Nakahira Y, Katayama M, Ishiura M, and Kondo T. Transcriptional regulation of the circadian clock operon kaiBC by upstream regions in cyanobacteria. Mol Microbiol 2005 Sep; 57(5) doi: /j x pmid: Wang J. Recent cyanobacterial Kai protein structures suggest a rotary clock. Structure 2005 May; 13(5) doi: /j.str pmid: PubMed HubMed [1] 6. Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, and Kondo T. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 2005 Apr 15; 308(5720) doi: /science pmid: PubMed HubMed [2] 7. Imai K, Nishiwaki T, Kondo T, and Iwasaki H. Circadian rhythms in the synthesis and degradation of a master clock protein KaiC in cyanobacteria. J Biol Chem 2004 Aug 27; 279(35) doi: /jbc.M pmid: PubMed HubMed [3] 8. Naef F. Circadian clocks go in vitro: purely post-translational oscillators in cyanobacteria. Mol Syst Biol 2005; doi: /msb pmid: