Promoter P_hs

Promoter P_hs is a novel thermal response designed by Wayne E Taylor et. al; it is super sensitive to temperature increase. We would expect to see the sudden increase in expression of the reporter gene, in this case, mRFP. Before start, we incubate the E. coli with promoter P_hs and mRFP as a reporter at room temperature 25 Celsius degree. We transient shift the E. coli to a higher temperature ambient (37 Celsius degree), and closely monitor the expression of the reporter. As we can see in the figure, the promoter responds to the temperature changes pretty quick and after 30 minutes, it show 6 fold augmentation in mRFP emission (wavelength 610 nm). We continue the test and tendency of increment shows no sign of subsidence.

Modified P_CI

The circuit incorporates a temperature sensitive cI promoter(CIts) to sense the temperature upshift. In our test we use a thermal adjustable plate reader to detect the mRFP flurorescence. In the beginning, we keep the temperature under 30 Celsius degrees for over 1 hour, and then detect the emission of mRFP. As the dimerized CIts repressor in lower temperature would specifically bind and repress P_CI, and further hinder the expression of mRFP. We can expect the emission to be low under 610 nm wavelength. We then abruptly increase our temperature to 37 celsius degrees, as we expect the CIts dimer would decompose and lose the function of repressing mRFP expression.

The result showed a low level of mRFP expression under wavelength of 580 nm (excitation) and 610 nm (emission). After the sudden temperature upshift, the expression of mRFP steadily rises, and results in 5.7 folds increase in the 8th hour after the temperature upshift. This proved the fact that repressor CIts and P_CI can largely lead to increase in protein expression, and can be used in our circuit design as it may turn on the circuit inside human body and spontaneously close down after the bacteria is expelled out.