and could The little oscillator that could and

and could… The little oscillator that could. and could… http: //www. worldofbubble. com/thomas_tank_engine/thomas. html

• Some strains of cyanobacteria oscillate on a circadian cycle. This cycle is driven by the interaction of three proteins, Kai. ABC, 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 -60 hours by point mutations of Kai. C (Kondo et al. , 2000).

• Transcriptional repression system • • • T~200 min Is not stable over time Advantages of Cyanobacteria oscillator • • • Plasmid to right, GFP reporter ‘Lite’ means destruction tag Stable over time Potentially more robust due to evolutionary development Post translational mechanism means less energy? Problem with implementation in later generations According to Elowitz: • “However, the reliable performance of [cyanobacteria] circadian oscillators can be contrasted with the noisy, variable behavior of the repressilator…It would be interesting to see whether one could build an artificial analogue of the circadian clock. ”

Deliverable: Bacterial Nightlight in E. coli Fallback: Bacterial Nightlight in Cyanobacteria 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. http: //www. footvolley. net/images/ronaldo%20 world%20 cup%20 goal. jpg

Obtain an appropriate strand of cyanobacteria (1 -2 wks) 1. 2. 3. • • • 4. Contact MIT i. Gem team for leads Synechococcus PCC 7942 or WH 8102 Extract the Kai. ABC genes from cyanobacteria and biobrick them (12 wks) Design of primers can be done beforehand Design a feasible E. coli sequence for Kai. ABC, and synthesize it (can be done in parallel with step 1) (1 -2 wks) Research the modifications we will need to make to the cyanobacteria genes to make them compatible with E. Coli; if they’re small, we won’t need to synthesize the whole sequence. Instead of synthesizing entire 3 kb sequence, break into smaller sequences to be synthesized separately to save on cost, and recombine by PCR. Insert both sequences (synthesized and Bio. Brick’d from cyanobacteria) into E. Coli and test (5+ wks)

http: //www. compendia. co. uk/acatalog/risk. jpg � 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 � Other environmental factors in E. coli may hinder the oscillator � More proteins may be involved than Kai. ABC � But Kai. ABC have been shown to work in vitro � Problems with synthesis of Kai. ABC � Not obtaining the cyanobacteria from various sources � This can be resolved by using alternative methods of synthesis � Testing � Test three plasmids attached to Kai. A, Kai. B, and Kai. C � Loss of time, effort, and resources � Resolved by implementing intermediate 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 Kai. C phosphorylation using antibody staining. But this doesn’t help us make the cell do any useful work.

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 14 h to 60 h (Kondo et. al 2000) with Kai. C 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. i. Gem Performance: A robust Biobricked oscillator, and its application in our system, will impress i. Gem 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.