Figure 1: Design of the system to test dually repressible promoters. Each promoter is composed of at least two different operators (in blue) recognized by specific repressors and regulates the expression of a reporter gene (mRFP1 in red). A - Only one copy of each repressor is present downstream of the promoter core. B - A copy of one of the two different operators is present upstream of the promoter core to possibly enable a better control of gene expression.

Part number	Part Name	Sequence	Size
BBa_K2510018	T7 - O-HKcI - O-P22c2 promoter	GTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGATTTAAGTGTTCTTTAATCGCTGTTCCGCTG	74
BBa_K2510019	T7 - O-HKcI - O-TetR promoter	GTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGTCCCTATCAGTGATAGAGATCACACTCCTTC	74
BBa_K2510020	T7 - O-P22c2 - O-HKcI promoter	GTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTGAACCATAAGTTCAGCTCTG	74
BBa_K2510021	T7 - O-P22c2 - O-TetR promoter	GTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTCCCTATCAGTGATAGAGATCACACTCCTTC	84
BBa_K2510022	T7- O-TetR - O-HKcI promoter	GTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATCACACTCCTTCTGAACCATAAGTTCAGCTCTG	74
BBa_K2510023	T7 - O-TetR - O-P22c2 promoter	GTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATCACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG	84

Testing of the promoters was performed in vivo in E. coli BL21 (AI) and E. coli BL21 (DE3). Both strains were tested to counter the effect of the T7 polymerase and at least one of the repressors being regulated by the same promoter. Analysis of fluorescence using a flow cytometer, testing four conditions for each strain: no inducer, each inducer indivudually - IPTG and arabinose - and both inducers together. The testing was performed on strains containing or not the repressors gene. The output measured is mRFP1 fluorescence and inputs ,i.e. repressor concentrations, are approximated by the concentration in fluorescent reporter protein - eyfp or ecfp. All florescences were measured using a flow cytometer.

The analysis of the flow cytometry data, obtained in the different conditions shows that

We wanted to combine two aspects of our project: our designed logic gate and the photocaged repressors. We chose to test them in a cell-free environment for both practicality reasons and to show that every aspect of our project was compatible with a cell-free environment. Using the NEB PURExpress kit for protein expression, we tested the impact of repressor caging on promoter activity. Violet light leads to the monomerization of Dronpa, so it releases the repressor, whereas Cyen light should leads to its dimerization, i.e. the caging of the repressor.

We expected to observe no red fluorescence after exposure to violet light and fluorescence after exposure to cyan light, as we thought the system would work similarly to a non-caged repressor. However, it appears that the fluorescence levels are increased over 1000-fold when the repressor is not caged.

One explanation for this behavior could be that the dimerization of Dronpa, instead of preventing repressor binding to its operator, actually makes it's binding more permanent, thus leading to the lower fluorescence level.