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Explore the potential of constructing a bacterial nightlight using cyanobacteria oscillation as the basis. Learn about KaiABC proteins, their transcriptional repression system, and the stability of cyanobacteria oscillators. Discover intermediate goals and challenges in creating a functional oscillator Biobrick. Find solutions for codon bias, protein involvement, and integration with E. coli. Dive into the journey of synthesizing KaiABC and testing plasmids to overcome obstacles and achieve success.
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and could… and could… The little oscillator that could. and 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, 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 • 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.”
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 http://www.footvolley.net/images/ronaldo%20world%20cup%20goal.jpg
Obtain an appropriate strand of cyanobacteria (1-2 wks) • Contact MIT iGem team for leads • Synechococcus PCC7942 or WH8102 • Extract the KaiABC genes from cyanobacteria and biobrick them (1-2 wks) • Design of primers can be done beforehand • Design a feasible E. coli sequence for KaiABC, 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 3kb sequence, break into smaller sequences to be synthesized separately to save on cost, and recombine by PCR. • Insert both sequences (synthesized and BioBrick’d 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 Other environmental factors in E. coli may hinder the oscillator More proteins may be involved than KaiABC But KaiABC have been shown to work in vitro Problems with synthesis of KaiABC Not obtaining the cyanobacteria from various sources This can be resolved by using alternative methods of synthesis Testing Test three plasmids attached to KaiA, KaiB, and KaiC Loss of time, effort, and resources Resolved by implementing intermediate goals such as a "nightlight" in cyanobacteria, not E. coli http://www.compendia.co.uk/acatalog/risk.jpg
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 doesn’t help us make the cell do any useful work.