Difference between revisions of "Part:BBa K2100000:Experience"

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Applications of BBa_K2100000

We used our synthetic promoter to build multiple constructs including: - pERE3:eYFP to characterize its base level functionality across the cell lines - pERE3:TALER14 and pERE3:BM3R1 to characterize the cascade of our promoter with different repressors - pERE3:TP901 to characterize the cascade of our promoter with the serine recombinase

First, we characterized the synthetic ERE3 promoter in three cell lines: MCF-7, ISH, 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 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 to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 50 nM E2.

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 additionally ran this experiment in ISH cells. However, the transfection efficiency was not high enough to determine conclusive results about the functionality of pERE3:eYFP in

User Reviews

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 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 .05 nM, .1 nM, .25 nM, .5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM.
+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.
 https://static.igem.org/mediawiki/2016/6/6d/T--MIT--khb_plot_of_e3_forexperience.jpeg
-However this experiment revealed that our concentrations must have already been at saturation because there was no stratification between all of the different estrogen concentrations.
+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.
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