Difference between revisions of "Part:BBa K2100042:Experience"

Revision as of 02:59, 20 October 2016

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Applications of BBa_K2100042

Cascade of pERE3:BM3R1

We experimented with our promoter pENTR pERE3 cloned with a repressor (pENTER BM3R1) to test the functionality of the promoter in a cascade. This experiment entailed multiple plasmids to be activated: pERE3:BM3R1 (500ng), pBM3R1:EYFP (170ng), hEF1a:mKate (170ng), hEF1a:Gal4VP16 (170ng), which is approximately a 3:1:1:1 ratio of plasmids.

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.

The first cell line in which we deployed our genetic circuit was ISH, the endometrial epithelial cell line. We had expected eYFP expression to decrease after induction of our promoter - repressor cascades with E2.

[ISH image here]

Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.

However, we were unable to resolve a clear fold difference between the uninduced and induced population in the pERE3 and BM3R1 cascade. 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.

We next proceeded to deploy this experiment in MCF7. We hypothesized that we were unable to resolve a clear fold difference in our pERE3 - BM3R1 cascade 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.

[MCF-7 image here]

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 the pERE3-BM3R1 cascade. Given more time, we would like to explore whether transfecting our entire circuit on one plasmid instead of four separate plasmids would lead to better results.

User Reviews

UNIQ0364775a20bcbde6-partinfo-00000000-QINU UNIQ0364775a20bcbde6-partinfo-00000001-QINU

@@ Line 5: / Line 5: @@
 ===Applications of BBa_K2100042===
+<br><br>
+<b><center>
+Cascade of pERE3:BM3R1
+</center></b><br><br>
+We experimented with our promoter pENTR pERE3 cloned with a repressor (pENTER BM3R1) to test the functionality of the promoter in a cascade. This experiment entailed multiple plasmids to be activated: pERE3:BM3R1 (500ng), pBM3R1:EYFP (170ng), hEF1a:mKate (170ng), hEF1a:Gal4VP16 (170ng), which is approximately a 3:1:1:1 ratio of plasmids.
+https://static.igem.org/mediawiki/2016/thumb/5/56/T--MIT--khb_repressors_for_parts.jpeg/778px-T--MIT--khb_repressors_for_parts.jpeg
+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.
+The first cell line in which we deployed our genetic circuit was ISH, the endometrial epithelial cell line. We had expected eYFP expression to decrease after induction of our promoter - repressor cascades with E2.
+[ISH image here]
+Blue contours represent the cell population that was left uninduced, green contours represent the cell population that was induced with 5 nM E2.
+However, we were unable to resolve a clear fold difference between the uninduced and induced population in the pERE3 and BM3R1 cascade. 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.
+We next proceeded to deploy this experiment in MCF7. We hypothesized that we were unable to resolve a clear fold difference in our pERE3 - BM3R1 cascade 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.
+[MCF-7 image here]
+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 the pERE3-BM3R1 cascade. Given more time, we would like to explore whether transfecting our entire circuit on one plasmid instead of four separate plasmids would lead to better results.
 ===User Reviews===