Difference between revisions of "Team:MIT/Experiments/Promoters/Experiment-Details-for-Cascades"
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<center><h1 style="background-color:#F20253;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Estrogen Sensitive Promoters in Repressor Cacades</h1> </center> | <center><h1 style="background-color:#F20253;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Estrogen Sensitive Promoters in Repressor Cacades</h1> </center> | ||
| − | We began testing how our estrogen responsive promoters behave in larger genetic circuits by first testing estrogen repressor cascades. We considered three repressors, BM3R1, TAL14, TAL21. | + | We began testing how our estrogen responsive promoters behave in larger genetic circuits by first testing estrogen sensitive promoter - repressor cascades. We considered three repressors, BM3R1, TAL14, TAL21. |
<u><b><h2>Our Genetic Circuit for Repressor Cascade Characterization </h2></b></u> | <u><b><h2>Our Genetic Circuit for Repressor Cascade Characterization </h2></b></u> | ||
Revision as of 03:04, 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. Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.
