Difference between revisions of "Team:MIT/Experiments/Promoters/Experiment-Details"
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| − | <img src= "https://static.igem.org/mediawiki/2016/c/c6/T--MIT--MCF-7pERE3.jpg" alt = 'Fluorescent reporter for transfection vs. reporter for promoter activity' style="width:400px;height:400px; float:left;" margin: 0 1.5%; class="rotate90"> | + | <img src= "https://static.igem.org/mediawiki/2016/c/c6/T--MIT--MCF-7pERE3.jpg" alt = 'Fluorescent reporter for transfection vs. reporter for promoter activity' style="width:400px;height:400px; float:left;" margin: 0 1.5%; class="rotate90"><p style="font-family: Verdana; float:left;"> The induced MCF-7 cells show up to a 10 fold difference in eYFP fluorescent output compared with the uninduced cells. However, there is not a defined fold difference in fluorescent output between the varying concentrations of estrogen induced. |
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| − | <p style="font-family: Verdana; float:left;"> The induced MCF-7 cells show up to a 10 fold difference in eYFP fluorescent output compared with the uninduced cells. However, there is not a defined fold difference in fluorescent output between the varying concentrations of estrogen induced. | + | |
Bar graph: The concentration of estrogen that stimulated the highest level of promoter activity was 0.05 nM, whereas the concentration that stimulated the lowest level of activity was 0.25 nM. These results are inconsistent with the hypothesis that increasing estrogen concentration in the cells will increase promoter activity, especially because the results from the 0.05 nM E2 well and the 10 nM E2 well are just about equal. Through a little research, we've concluded that the lack of staggered results is due to the fact that we dissolved E2 in ethanol. According to Etique et. al [1], MCF-7 cells grown in an ethanol medium were correlated with increased proliferation, ERalpha content, and ER transcriptional activity. We tweaked our experimental design for future experiments to contain a vehicle control which accounts for the proliferation and increase in ERalpha caused by ethanol. Then we can more accurately compare our promoters' basal levels with induced levels.</p> | Bar graph: The concentration of estrogen that stimulated the highest level of promoter activity was 0.05 nM, whereas the concentration that stimulated the lowest level of activity was 0.25 nM. These results are inconsistent with the hypothesis that increasing estrogen concentration in the cells will increase promoter activity, especially because the results from the 0.05 nM E2 well and the 10 nM E2 well are just about equal. Through a little research, we've concluded that the lack of staggered results is due to the fact that we dissolved E2 in ethanol. According to Etique et. al [1], MCF-7 cells grown in an ethanol medium were correlated with increased proliferation, ERalpha content, and ER transcriptional activity. We tweaked our experimental design for future experiments to contain a vehicle control which accounts for the proliferation and increase in ERalpha caused by ethanol. Then we can more accurately compare our promoters' basal levels with induced levels.</p> | ||
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Revision as of 03:01, 8 October 2016
Results from testing pERE promoters in MCF-7
The induced MCF-7 cells show up to a 10 fold difference in eYFP fluorescent output compared with the uninduced cells. However, there is not a defined fold difference in fluorescent output between the varying concentrations of estrogen induced. Bar graph: The concentration of estrogen that stimulated the highest level of promoter activity was 0.05 nM, whereas the concentration that stimulated the lowest level of activity was 0.25 nM. These results are inconsistent with the hypothesis that increasing estrogen concentration in the cells will increase promoter activity, especially because the results from the 0.05 nM E2 well and the 10 nM E2 well are just about equal. Through a little research, we've concluded that the lack of staggered results is due to the fact that we dissolved E2 in ethanol. According to Etique et. al [1], MCF-7 cells grown in an ethanol medium were correlated with increased proliferation, ERalpha content, and ER transcriptional activity. We tweaked our experimental design for future experiments to contain a vehicle control which accounts for the proliferation and increase in ERalpha caused by ethanol. Then we can more accurately compare our promoters' basal levels with induced levels.
Results from testing pERE promoters in ISH
The pERE3 promoter showed very little to no fold difference between those transfected into induced and uninduced cells. Qualitatively, the fluorescent output seems to have a bit of a staggered result based on the nM amount of E2 in the cell, but not significant enough to come to a solid conclusion about E2 concentration vs. promoter activity.
The pERE5 promoter showed essentially zero fold difference between those transfected into induced and uninduced cells. The fluorescent output also varied extremely little between different E2 amounts induced.
As the previous two promoters, the pERE6 promoter experiment produced zero/negligible fold difference between the fluorescent output of the uniduced vs the induced cells.
Transfection efficiency is around 1%. Since we had an extremely low portion of transfected cells, our data was not very representative of the cell population of ISH we cultured in our lab. Though we got relatively smooth line graphs due to high cell density, there are still a relatively low number of transfected events to analyze from cytometry. This makes our data set less robust than we want it to be.
Overall Conclusion
None of the promoters showed any sign of functioning in ISH cells, due to the lack of change in eYFP fluorescent output between induced and induced cells transfected with the pEREx-eYFP construct. Additionally, the fine sweep of E2 concentrations showed little to no variation in fluorescent output, but if the promoters aren't functioning properly (perhaps because their basal expression is ISH is very high), then increasing the concentration of E2 by small factors is not expected to have a huge impact on fluorescent output.
