Part:BBa_K332021:Design

This part K332021, added by other translation units and terminator at last, can be a new part which is negative controlled by protein tetR. In our work, we would then put RBS, luxI, double ter sites behind K332022, making it a complete functional unit.

In this portion of the design, we aim to achieve population control by combining a cell-signaling system (luxR/luxI) and suicidal gene for controlling the number of E.coli. The population controlling function in the engineered E.coli can warn us of high-density E. coli congregations by emitting red fluorescent that signals E.coli overpopulation.

LuxR/luxI is often used as a self-regulation system. When E.coli are growing, a metabolite, SAM, are produced. LuxI can then transfer SAM to another compound AHL. Finally, luxR combine with AHL to activate Plux. When the population of E.coli grow, luxR are more likely to combine with AHL, so Plux is getting more efficient. We can use this property to put a part behind Plux, the concentration of that part would rise as more E.coli propagates.

In our device, we put ccdb behind Plux, it is a protein that can cause E.coli to die. As mentioned above, more ccdb could be produced as the population of E.coli grow, that is, E.coli tend to die when they are getting more and more. Finally, combining all these parts makes a self-population-controled E.coli.

Modeling of our work:

Population control system consists of three genes: LacI, LuxR, and ccdB. The initial population size of Terminator is the input to the system. The concentrations of LacI, LuxR, and ccdB proteins and bacterial population are the outputs. This system can be modeled by differential equations as follows.

Alpha-C, D and bac are production rates of the corresponding targets, which are assumed to be given constants. Gamma-LuxR, gamma-ccdB, gamma-AHL and gamma-bac are decay rates of the corresponding targets. For an activator AHL/LuxR complex, Hill function is an S-shaped curve which can be described in the form x^n / (K +x^n). K is the activation coefficient and n determines the steepness of the input function (Alon, 2007). The k2 is the production rate of AHL that synthesized by the LuxI protein. For an inhibition of bacterial population, Hill function can be described in the form 1 / (1 +x^n). Because many of the in vivo rates of the biochemical reactions we simulated are unknown, the values of the kinetic parameters used in the simulation were initially obtained from the literature and educated guesses. From the gene network design procedure, initial values and numerical configurations were given, which lead to several rounds of simulation and results. (Fig.2).

Fig.4: The simulation result of the population control system. When the population size of E. coli rises to the threshold level, ccdB proteins are induced by AHL to restrict the population size.

At present, we have satisfactory in-silico results. The next step is to implement the “population control system” into the genetic circuit and ultimately the host bacteria for experimental data.

Source

all from IGEM's gene bank

BBa_R0040 comes from 2009 Kit Plate 1, well 6I

BBa_B0034 + BBa_C0062 comes from BBa_J37033, 2009 Kit Plate 2, well 4O