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"><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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<img src= "https://static.igem.org/mediawiki/2016/c/c6/T--MIT--MCF-7_pERE3.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.</p>
  
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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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<img src= "https://static.igem.org/mediawiki/2016/c/c6/T--MIT--MCF-7_pERE3_Bargraph.jpg" alt = 'Fluorescent reporter for transfection vs. reporter for promoter activity' style="width:400px;height:400px; float:left;"  margin: 0 1.5%; class="rotate90">
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<p style="font-family: Verdana; float:left;">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 03:10, 8 October 2016

Promoter Characterization Experiments in MCF-7, ISH, and tHESC

Results from testing pERE promoters in MCF-7



Fluorescent reporter for transfection vs. reporter for promoter activity

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.

Fluorescent reporter for transfection vs. reporter for promoter activity

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.