Part:BBa_K2100039:Experience
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Applications of BBa_K2100039
This cascade of pBM3R1:eYFP was in the testing of the BM3R1 repressor. We used this promoter cascaded with eYFP as an indicator as to how much repression is caused by the actual repressor when activated. Here are the experiments we did using the BM3R1 repressor and its partnering promoter.
We used our repressor to build multiple constructs including:
- pTRE:BM3R1 to characterize its base level functionality
- pERE3:BM3R1, pERE5:BM3R1 and pERE6:BM3R1 to characterize the cascade of our promoters with the BM3R1 repressors
We characterized the repressor BM3R1 by creating a plasmid TRE:BM3R1, containing BM3R1 under the control of a doxycycline inducible promoter, TRE. This allowed for us to modulate the repressor expression by varying the concentrations of doxycycline added. We also created a repressible promoter controlling the expression of a fluorescent protein. This plasmid allowed for us to measure the repression occurring.
In order for us to analyze the level of repression, we need to see sustained presence. Upon increasing the concentration of doxycycline, we were unable to see that sustained presence of the repressors. From the results of testing BM3R1 below, it can be seen that although there is activation early on, the production of repressor falls at 500 nM. This could possible be due to errors in doxycycline dilutions at 500 nM and above.
Next we experimented with our promoter pENTR pERExN cloned with repressors (pENTER BM3R1) to test the functionality of the promoters in a cascade. These experiments entailed multiple plasmids to be activated: pERExN:BM3R1 (500ng), pBM3R1:EYFP (170ng), hEF1a:mKate (170ng), hEF1a:Gal4VP16 (170ng), which is approximately a 3:1:1:1 ratio of plasmids.
(1) Estrogen diffuses into the cell and binds with the estrogen receptor. (2) Estrogen receptors will homodimerize with one another forming an activation complex. (3) Estrogen receptor will bind to our synthetic promoter (4) Production of repressor protein (5) Repressor binds to binding sites upstream of an eYFP reporter (6) Transactivator Gal4-VP16 is constitutively produced (7) Gal4-VP16 binds to sites on pRep (8) eYFP is produced as readout depending upon how active repressors are (9) Constituively active transfection marker hEF1a:mKate allows us to bin and analyze the data.
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.
Experiment with pERExN:BM3R1 in ISH:
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.
This is pERE3:BM3R1 in ISH.
This is pERE5:BM3R1 in ISH.
This is pERE6:BM3R1 in ISH.
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 any of the pERExN and 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.
Experiment with pERExN:BM3R1 in MCF7:
We next proceeded to deploy this experiment in MCF7. We hypothesized that we were unable to resolve a clear fold difference in our pERExN - 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.
This is pERE3:BM3R1 in MCF7.
This is pERE5:BM3R1 in MCF7.
This is pERE6:BM3R1 in 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 pERExN and BM3R1 cascades due to a poor transfection efficiency in the experiments.
Overall, we were still unable to resolve a clear fold difference between the uninduced and induced population in any of the pERExN or TRE and 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.
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