This gene circuit is designed to produce a functional protein in response to a particular environmental signal. Since the dose response curve for different regulators and the expression intensity of a certain promoter may affect the performance of the circuit, the initial version of the circuit is very likely to be non-functional. For example, the expression level of the regulator gene may be too strong and the circuit is always active even without induction. Or, the regulator binding might be too strong to the promoter, making the expression level of the protein very low even when desired. Either way, it is impossible to predict the best solution for your design. To optimize the circuit, the customer wanted to use promoters with different strength to fine tune the expression level of three genes. They chose five promoters with strengths from very weak to very strong to express the genes. In addition, they want to test different versions of the sensors and regulators with different kinetic properties to optimize the circuits.
A client designed a gene circuit for producing a functional protein upon sensing specific environmental signals. Two variations were selected for each of the three genes in the pathway and five promoters with strength levels from very weak to very strong were selected for each gene, making the theoretical combination 1,000.
Each of the 1,000 constructs were assembled individually. Each plasmid is then sequence verified, followed by transformation of plasmids into appropriate expression host to test performance.
(A) Response curve after induction. The optimized circuit exhibited stronger and more prompt response behavior than the initial. (B) Inducer dose response for promoter-3 strength. The medium-strength promoter-3 showed the best dose-response behavior.