Part:BBa_K2100019:Experience
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Applications of BBa_K2100019
We used our recombinase to build multiple constructs including:
- pEGSH:TP901 to characterize its base level functionality
- pERE5:TP901 and pERE6:TP901 to characterize the cascade of our synthetic promoters with a serine recombinase
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.
Also, we experimented with our promoters pENTR pERE5, pERE6 cloned with a serine recombinase to test the functionality of the promoters in a cascade. These experiments entailed multiple plasmids to be activated: pERE5/6:TP901, hEF1a:Flipped_eYFP-recombinase sites, and a transfection marker, hEF1a:BFP. Upon activation of TP901, the inverted eYFP gene flanked by recombinase recognition sites is flipped to the correct orientation and expresses fluorescence. We tested these constructs in the cell line MCF7.
We have previously demonstrated activation of TP901 under the inducible promoter EGSH, which is activated by transactivator VgEcR along with estrogen analog PonA. Despite high levels of TP901 basal expression, we observed a clear difference in activation between the induced and uninduced wells. We expected to see similar results in this experiment with TP901 under estrogen inducible promoters.
For our transfection experiment, we induced half the wells with 5.0 nM E2 in order to compare on vs. off states of the promoter.
This image shows the differences between pERE5:TP901 when uninduced and induced with E2.
This image shows the differences between pERE6:TP901 when uninduced and induced with E2.
Unfortunately, we had poor transfection efficiency in this experiment, and thus the results are inconclusive. The data showed no clear fold difference between the induced and uninduced populations. We would like to try this experiment again in the future to get better results.
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