Part:BBa_K332022:Design
Plux + RBS + mRFP + double terminator
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 795
Illegal AgeI site found at 636
Illegal AgeI site found at 748 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The above picture show why we design K332022 and how we use it, in our complete work we add another suicide gene, ccdb, inside K332022. The mRFP proteins are produced after luxR and AHL are produced in E.coli and Plux is activated. When the population of E. coli reaches to a high level, meaning that the concentration of AHL and luxR also get higher, the Plux has a higher activation rate, resulting in more mRFP production and stronger red fluorescence.
With this circuit, we can observe the population of E.coli clearly-high when red fluorescence is strong, low on the other hand. Appearance of red fluorescence can also suggest us adopt some methods to control the density of group. Furthermore, more ccdb protein are produced when E.coli population get high, making E.coli tend to die. This means that the circuit has has the ability of population control.
Modeling and simulation
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).
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