Part:BBa_K2100028:Experience

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Applications of BBa_K2100028

pERE5:eYFP is a cascade of our synthetic promoter pERE5 with a yellow florescent tag, eYFP. The parts were combined through a gateway cloning technique called an LR reaction.

We characterized the construct pERE5:eYFP in three cell lines: ISH, 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 pERE5: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 11 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 pERE5: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 y axis measures yellow fluorescent output and the x axis measure red fluorescent output from the transfection marker. 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 pERE5: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 pERE5 in the tHESC cell line.

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.

Unfortunately, we did not observe a clear fold difference for pERE5 upon induction with 10 nM E2. We suspected that perhaps we had not induced the system with enough E2 and included 50 nM in our finer sweep of E2 levels.

We also attempted to demonstrate a graded response of our promoters to changing E2 levels in tHESC.

We induced cells transfected with pERE5 to either 2 nM, 10 nM, or 50 nM E2(represented by the color contours in the above plot) in an attempt to obtain graded eYFP production. However, we still did not observe a clear fold difference for pERE5:eYFP.

Experiment in ISH:

We additionally transfected ISH cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pERE5:eYFP (the same 1:1 ratio as the experiments ran in MCF-7 and tHESC) to examine the promoter's transcriptional activity increase when induced with estrogen.

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.

Our promoter pERE5 demonstrated a 3 fold increase in activity between the induced and uninduced populations. We suspect the differences in promoter activity between this transfection and those done in MCF7 are due to different basal levels of ER in the two different cell lines.

We also attempted to stratify the response of our promoter to varying levels of estrogen in ISH. We sought to obtain a finer characterization through exposing transfected cells to a sweep of E2 concentrations including 0.25 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM. Just like before, we had hypothesized that our promoters would demonstrate a graded response to estrogen induction.

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 just as seen with MCF7.

Overall, our promoter pERE5 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, pERE5, in multiple cell lines by quantifying the yellow florescence expression in comparison to the red florescence expression.

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