Part:BBa_K2100010:Experience

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Applications of BBa_K2100010

We used our promoter to build the pHybrid:eYFP construct to characterize the functionality of our promoter.

First, we characterized the synthetic pHybrid promoter in two cell lines: MCF7 and tHESC. All cell lines have endogeneous progesterone Receptor A and estrogen receptor alpha. We analyzed data from cells induced with either 1 uM of MPA or 10nM of E2 compared to those uninduced with hormones. This concentration of MPA, the progesterone analog, was recommended to us as an appropriate "on" or saturating concentration by the Griffith Lab which provided us with these cells. The estradiol(E2) is diluted and mixed with ethanol at small percents, so we also tested an ethanol vehicle to account for the proliferation the cells undergo after being induced.

We were unable to demonstrate dual sensing of both estrogen and progesterone in one cell line by our hybrid promoter (pHybrid) due to limited experimental time after completing its construction and cloning. The following characterization experiments in tHESC cells tested how the hybrid promoter responds to a 1 uM induction of progesterone (MPA) and how in MCF7 cells the hybrid promoter responds to a 10 nM induction of E2 (estradiol) in MCF-7 cells.

We transfected both cell lines with 250ng of hEF1a:mKate as a transfection marker and 250 ng pHybrid:eYFP to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced. 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 pHybrid.

First we transfected pHybrid promoter into MCF7, comparing the uninduced population to a population induced with 10 nM E2.

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 a 12 fold difference between the yellow fluorescent output of the uninduced MCF-7 cells and the ones induced with 10 nM E2.

We also transfected pHybrid promoter into tHESC, 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 an 100 fold difference between the yellow fluorescent output of the uninduced MCF-7 cells and the ones induced with 10 nM E2.

Overall, pHybrid demonstrated successful estrogen signaling sensing in MCF7 and successful progesterone signaling sensing in tHESC. With more time, we believe pHybrid could accurately sense both estrogen and progesterone simultaneously, and yield a large difference between on and off states.

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