Part:BBa_K2510028

This dually repressible promoter allows for the creation of a logic-gate, specifically a NOR-Gate. Indeed, gene expression will occur only when the two repressor are either expressed but non-functioning or absent from the system altogether. To design such a promoter, we combined insulated promoter that had been previously tested to form repressible promoter to create new dually repressible promoters that can be used to form a logic gate. The promoter was tested both in vivo and in a cell-free environment using repressors that were caged with a photoswitchable fluorescent protein.

In vivoTesting

Promoters were tested in vivo in 3 different strains of E. coli. The results show that the system is not as efficient as it could be, and insularity of promoter elements is still a long way off. Figure 5: Data obtained from flow cytometry experiments. The number showed is the mean of medians for 3 different experiments.

Cell-free testing

This synthetic operator was part of the library that we used to test the activity of repressors caged with Dronpa

Figure 1 : the experiment conducted with the repressors caged with Dronpa Figure 2 : An over view of the results of the cell-free experiment. Each promoter was tested with its cognate repressors. Our results show that in 90% of our constructs caging the repressors with Dronpa has increased the repression strength. We have also obtained a wide linear range of repression strength-indicated by the red slope-. Figure 3: detailed Results of the cell-free experiment. Each promoter was tested with its cognate repressors. Top: Testing with TetR caged with either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)Middle: Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated version(BBa_K2510113).Bottom: Testing with HKcI caged with either wt- Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111) Figure 4: the wtDronpa has a better control of HKC1 and C2P22 repressors while the mutDronpa has a better control of TetR repressors Sequence and Features BBa_K2510028 SequenceAndFeatures