Part:BBa_K2100026:Experience

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Applications of BBa_K2100026

We constructed the vector hEF1a:VgEcR with an LR reaction and used it to characterize the EGSH promoter in HEK293 cells. The mechanism involved in the activation of pEGSH is a VgECR complex bound to an RXR. We included constitutive expression of this construct in our experimental design ensure activation of pESGH when induced with PonA.

We used a 3:1:1 ratio of EGSH:mKate, transactivator hEF1a:VgEcR, and transfection marker hEF1a:BFP based on our investigation into the ideal DNA ratios for experiments involving EGSH induction. The 2014 MIT iGEM team, who also used EGSH as an inducible promoter, reported seeing the greatest success with this ratio. We induced cells containing these plasmids with 5 different amounts of PonA. We also added a control sample of HEK293 lacking the transactivator necessary for EGSH, to further characterize any leaky expression of the EGSH promoter. We used hEF1a-tagBFP as a transfection marker.

We observed a <2 fold increase between induced and uninduced EGSH, but we noticed a decent amount of basal expression with no PonA added. The saturation point occurred around 5 uM of PonA, the point at which adding more PonA does not significantly, if at all, increase red fluorescent output, so we determined that this concentration is the best amount of PonA to compare on/off states of the promoter.

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