Part:BBa_K2100019:Experience

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Applications of BBa_K2100019

We set out to characterize TP901 under an inducible promoter to determine the basal expression levels in order to determine what further repression systems we would need. We cotransfected the EGSH: TP901 and hEF1a: attB-flipped EYFP-attP expression vectors along with EGSH: mKate (to indicate how much TP901 the cells were expressing), hEF1a: VgEcR (the transactivator for EGSH), and hEF1a: BFP (a transfection marker) into HEK293 cells. We induced the cells with six different concentrations of PonA: 0 uM, 0.1 uM, 0.5 uM, 1 uM, 2 uM, and 5 uM.

Experimental Setup:

Results:

As shown in the graph, we saw approximately a two-fold difference in yellow fluoresence between the uninduced cells and the cells induced with 2 uM or 5 uM (which seemed to be at saturation). We observed a significant amount of basal activity of TP901 even in the absence of PonA, however, so we concluded that an inducible promoter is too leaky to silence the activity of TP901. This issue was our motivation for exploring the L7Ae/k-turn motif as a way to lower the basal expression of TP901, and we hope that this system will allow us to inhibit recombination when the system is uninduced but not when it is activated.

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