Part:BBa_K2100030:Experience

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Applications of BBa_K2100030

pPRE3:eYFP is a cascade of our synthetic promoter pPRE3 with a yellow florescent tag, eYFP. The parts were combined through a gateway cloning technique called an LR reaction.

We characterized the cascade of our synthetic promoter PRE3 with the eYFP gene in tHESC. All cell lines have endogeneous progesterone receptor A. We analyzed data from cells induced with 1 uM of MPA compared to those uninduced with hormones. This concentration of MPA was recommended to us as an appropriate "on" or saturating concentration by the Griffith Lab which provided us with these cells.

We transfected tHESC cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pPRE3:eYFP to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 1 uM MPA. This ratio was chosen to be 1:1 based on the small amount of plasmids being transfected. We were looking to deterimne the on-off functionality of our promoter pPRE3 in the tHESC cell line.

The panel above represents results for pPRE3:eYFP transfected into the tHESC cell line, comparing the uninduced population to a population induced with 1 uM MPA. The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.

The results showed little to no increase in fold difference of yellow fluorescence when comparing induced vs. uninduced cells. Unfortunately, we could not prove with this experiment that the PRE3 promoter increases activity in response to progesterone induction.

Overall, our promoter pPRE3 cascade with eYFP demonstrates no fold difference when induced with progesterone in multiple cell lines. This construct did allow us to characterize the exact activity of our synthetic promoter, pPRE3, in multiple cell lines by quantifying the yellow florescence expression in comparison to the red florescence expression.

Note: We attempted to characterize pPRE3 in MCF-7 cells as well, however due to low transfection efficiency we were unable to get conclusive results about the on-off functionality of the promoter.

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