Revision as of 01:04, 9 October 2016

Promoter Testing Workflow

Results from testing pERE promoters in MCF-7

pERE3

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.

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.

pERE5

The results show about a 2- to 4-fold increase in promoter activity when induced with estrogen. However, when comparing the estrogen fine sweep results, it appears that the lower concentrations of estrogen (0.05 nM, 0.25 nM) had increased fluorescent output levels from the promoter compared with the cells that were induced with higher concentration (5 nM, 10 nM). The promoter proves to be functionable, but not necessarily to the caliber we need for our project. Our project relies on estrogen responsive promoters that can sufficiently detect the difference between the phases of the menstrual cycle. A two to four fold increase in promoter activity and an illogical correlation between output and varying estrogen levels will not be very helpful in the context of our circuit. So, we must keep trying.

pERE6

The results show about a 2 fold difference between basal and induced promoter activity, with no evident correlation between output and increasing concentration of E2 in the cell. Similarly to the pERE5 promoter, the fold difference between the on/off states of the promoter might not be sufficient for our circuit to identify different phases of the menstrual cycle.

Notes of Interest

-In this experiment, our control was an uninduced well of cells transfected with the promoter-reporter construct. There was no vehicle control. Cell proliferation might partially be the cause increased fluorescent output.

-Transfection Efficiency is around 10-15%.

Overall Conclusion

The pERE3 promoter showed the greatest difference between off/on states out of all three promoters tested in MCF-7 cells. Based on these experimental results, we would choose the pERE3 promoter as the estrogen-responsive for our circuit. However, we still need to test these promoters in endometrial cell lines to best model conditions our circuit will be implemented in, and use a vehicle control to account for cell proliferation and greater ERalpha expression. This experiment was a good baseline test to see how our promoters worked, and we used it as a stepping stone to design future experiments and compare results with.