Part:BBa_K2100047:Experience

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Applications of BBa_K2100047

We experimented with our promoter pENTR pERE5 cloned with a serine recombinase, TP901, to test the functionality of the promoters in a cascade. These experiments entailed multiple plasmids to be activated: pERE5: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.

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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