Difference between revisions of "Team:MIT/Experiments/Promoters/Experiment-Details-for-Cascades"
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<p> We have previously demonstrated activation of TP901 under-promoter EGSH, which is activated by estrogen analog PonA. We expected to see similar results in this experiment, with more yellow fluorescence, indicating TP901 activation, in the wells induced with 5nM E2. </p> | <p> We have previously demonstrated activation of TP901 under-promoter EGSH, which is activated by estrogen analog PonA. We expected to see similar results in this experiment, with more yellow fluorescence, indicating TP901 activation, in the wells induced with 5nM E2. </p> | ||
| − | <center> <img src = "https://static.igem.org/mediawiki/2016/d/d6/T--MIT--pERE5-TP901.jpg"; alt = '' style="width:544px;height:599px;" margin: 0 1.5%;> </center> | + | <center> <img src = "https://static.igem.org/mediawiki/2016/d/d6/T--MIT--pERE5-TP901.jpg"; alt = '' style="width:544px;height:599px;" margin: 0 1.5%;> <img src = ""; alt = '' style="width:544px;height:599px;" margin: 0 1.5%;> </center> |
<p> Unfortunately, we did not have success with this transfection, and thus there was no clear fold difference between the induced and uninduced populations. We would like to try this experiment again given more time. </p> | <p> Unfortunately, we did not have success with this transfection, and thus there was no clear fold difference between the induced and uninduced populations. We would like to try this experiment again given more time. </p> | ||
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Revision as of 17:24, 15 October 2016
Estrogen Sensitive Promoters in Repressor Cacades
Our Genetic Circuit for Repressor Cascade Characterization
Figure . Our estrogen sensitive promoters respond to increases in E2 levels by producing more of the repressor. The repressors then bind to binding sites in a promoter upstream of fluorescent reporter eYFP. The constitutively active trans-activator Gal4-VP16 sets a large basal eYFP expression when there is no repressor, so that a measurable drop in signal can be observed when repressors are active. Constituvely active hEF1a mKate serves as a transfection marker by which we bin our data.
Repressor Cascades in ISH
The first cell line in which we deployed was the ISH, epithelial cell line. We had expected eYFP expression to decrease after induction of our promoter - repressor cascades with E2. However, we were unable to resolve a clear fold difference between the uninduced and induced population in any of the pERE#n and TAL14, BM3R1 cascades. This is probably an artifact of poor transfection in the ISH cell line for this experiment (less than 2 percent transfected after cationic lipid transfection), which leads to erratic jumps in the data after binning by constitutive marker. In the future, we may want to try other modes of transfection for ISH to improve the transfection efficiency.
Figure . Results from pERE#n - repressor cascades pairing pERE3, pERE5, pERE6 and TAL14, BM3R1 transfected into MCF7. Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.
Repressor Cascades in MCF7
We hypothesized that we were unable to resolve a clear fold difference in our pERE#n - repressor cascades transfected into ISH because of the limited functionality of our promoters in the ISH cell line. So, we proceeded to transfect our cells into the MCF7 cell line where we had observed up to a 11 fold difference in the activity of some of our promoters.
Figure . Results from pERE#n - repressor cascades pairing pERE3, pERE5, pERE6 and TAL14, BM3R1 transfected into MCF7. Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.
Similarly, we had expected eYFP expression to decrease after induction of our promoter - repressor cascades with E2. However, we were still unable to resolve a clear fold difference between the uninduced and induced population in any of the pERE#n and TAL14, BM3R1 cascades. Given more time, we would like to explore whether transfecting our entire circuit on one plasmid instead of five separate plasmids would lead to better results.
Estrogen Sensitive Promoters in Recombinase Cacade
We also tested activation of recombinase TP901 under estrogen responsive promoters pERE5 and pERE6 in the MCF7 cell line. Upon activation of TP901, the inverted eYFP gene flanked by recombinase recognition sites is flipped and expresses fluorescence.
We have previously demonstrated activation of TP901 under-promoter EGSH, which is activated by estrogen analog PonA. We expected to see similar results in this experiment, with more yellow fluorescence, indicating TP901 activation, in the wells induced with 5nM E2.
Unfortunately, we did not have success with this transfection, and thus there was no clear fold difference between the induced and uninduced populations. We would like to try this experiment again given more time.
