Part:BBa_K2100027:Experience

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Applications of BBa_K2100027

pERE3:eYFP is a cascade of our synthetic promoter pERE3 with a yellow florescent tag, eYFP. The parts were combined through a gateway cloning technique called an LR reaction.

We characterized the construct ERE3:eYFP in two cell lines: MCF-7 and tHESC. All cell lines have endogeneous Estrogen Receptor alpha. We analyzed data from cells induced with estradiol (E2) and uninduced as a control. The estradiol 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.

Experiment in MCF-7:

We transfected MCF-7 cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pERE3:eYFP to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 5 nM E2. This ratio was chosen to be 1:1 based on the small amount of plasmids being transfected.

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. Since transient transfection results in an uneven distribution of plasmids, it is important to bin our data by transfection marker so that cells which received roughly the same number of plasmids can be compared against one another.

The results show an 8 fold difference in yellow fluorescent output between the induced MCF-7 cells and the uninduced cells, which improves on the results seen in Klinge et al. [1] for three estrogen responsive elements.

Additionally, in MCF7 we attempted to stratify the amount of activation of our promoter based on the amount of estrogen used to induce the cells. We ran an experiment where we kept the previously mentioned 1:1 ratio of transfection marker to pERE3:eYFP, but induced with varying levels of estrogen at .25 nM, .5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM. We had hypothesized that our promoters would demonstrate a graded response in eYFP production to this graded induction of E2 levels.

For the plot above, the colored contours represent different levels of E2 induction ranging from 0.25 nM to 10 nM. The pink contour in each graph represents the uninduced population. We did not observe a graded response in eYFP production in response to the sweep of E2 induction, instead observing saturation at 0.25 nM E2. We hypothesize that, because MCF7 overexpresses the estrogen receptor, relatively small E2 signals can still be transduced to large responses.

Our promoters were able to successfully sense changes in estrogen signaling in the MCF7 cell line. All three promoters demonstrate a fold increase of different magnitude upon exposure to estrogen. We have not yet been able to demonstrate a graded response of our promoters to changing E2 levels in MCF7. Instead we observed saturation at our lowest concentration tested, .25 nM.

Experiment in tHESC:

We transfected tHESC cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pERE3:eYFP (the same 1:1 ratio as the experiments ran in MCF-7) to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 50 nM E2. This was to test the on-off functionality of our promoter pERE3 in the tHESC cell line.

Results for our induction of pERE3 in tHESC as compared to an EtOH Vehicle Control. 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 show a 9 fold difference in yellow fluorescent output between the induced tHESC cells and the uninduced tHESC cells with a vehicle control (0.4% EtOH) to account for the possible increase in proliferation cells undergo when being induced with E2 dissolved in ethanol.

We have not yet been able to demonstrate a graded response of our promoters to changing E2 levels in tHESC.

Overall, our promoter pERE3 cascade with eYFP demonstrates extremely successful fold differences when induced with estrogen in multiple cell lines. This construct allowed us to characterize the exact activity of our synthetic promoter, pERE3, in multiple cell lines by quantifying the yellow florescence expression in comparison to the red florescence expression.

Note: We attempted to characterize pERE3:eYFP in ISH 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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