Repressilator is a tripartite device consisting of 3 repressors that can form a closed negative feedback loop and generate periodically oscillation. It is artificially designed and put into E.coli for functional tests which was proved for successful oscillation in vivo. Well-defined repressilator is thought to possess the precision that rivals nature systems which can be put into various practical application.
Our manipulations on repressilator are as follows:
1) We want to determine the period of solitary repressilator or coupled one or in different strains in our laboratory condition via triple reporters on utilized plasmid pLPT107.
2) IPTG is added to primarily synchronized the colony and we planned to verify this effect.
3) Instead of inviting engineered E.coli into living organism, we propose to validate the integration condition of engineered E.coli into artificial intestinal juice.
4) Build clpXP knock-out MC4100 strain to guarantee more stable oscillation period.
1) Constructed bacteria strains obeyed a 2 hour period in experimental condition.
2) iPTG addition could help direct a more robust synchronization in colony by amplify the oscillating amplitude.
3) Engineered strains could colonize stably in artificial intestinal fluid and the oscillation pattern kept uniformity as in culture media.
4) We successfully knocked out clpXP gene in position, however residual of pKD46 made our modified strain fail to select for ampicillin resistance of pLPT107.
Quorum sensing (QS) is a well-known natural system of prokaryotes in order to sense the population density with in a community. Recently, increasing researches begin paying close attention to QS system for extended use. QS can build connections among population in not only quorum density but also other information of intracellular status if coupled with signals from another system, which makes it a feasible and convenient tool for intercellular communication.
Quorum sensing is first added into our circuit to solve three main problems:
1) synchronize the repressilator coherently to achieve a precise population scale oscillation.
2) send and modulate periodic signal with enzyme (COMT) expression and coupling melatonin synthesis.
1) Optimize the modelling work in 2000 and complete design 4p0, 3p1 system.
2) Confirm quorum sensing in our circuit.
3) Successfully build the represillator-combined QS circuit. And cell B in 4p0 system.
Modeling results of repressilator indicated that faster translation is helpful to the period elongation, which is necessary to mimic the process of melatonin production in human. Translation initiation is the rate-limiting step of translation. Its rate is determined by multiple molecular interactions, including the hybridization of the 16S rRNA to the RBS sequence. Thus, we can change the translation rate via the modification of RBS.
1) We want to improve the translation rate of the original device (BBa_I13521) by modifing its ribosome binding site.
2) We want to confirm the methods of RBS prediction and plasmid construction could be credible.
1) We have designed 3 synthetic RBS with different strength by RBS Calculator.
2) We have replaced the RBS in the original reporter part (BBa_I13521).
3) We have measured the strength of our designed RBS, and confirmed the increase of translation rate.
4) We have confirmed that the methods we used were credible.