Team:Mines/Contribution
Characterization of Cadmium Sensing promoter Part#BBa_K1724000
Introduction
The CadA oromoter is a cadmium sensitive promoter. Its repressor is MerR. In high concentrations of Cadmium, the Cd2+ can rapidly bind with MerR and remove its inhibition of the CadA promoter. To characterize this part, we transformed the vector into E. coli and exposed it to varying levels of cadmium and measured red florescence protein levels.
Experimental Design
Well 7H of well plate 5 was rehydrolised and transformed into E. coli. The E. coli was then spread on LB/chloramphenicol plates and left to incubate at 37C overnight. A resulting colony was then taken from the plate and resuspended in LB/chloramphenicol solution, again overnight, at 37C and shaken at 250 rpm. 25 alicuots of the culture were taken at 1:20 dilutions. An OD600 measurement was taken and used to dilute to 25 0.1 OD600 colonys. After dilution, colonys were incubated for an hour at 37C. 100 ul of growth media and cadmium from diluted stock with colony alicuots was produced for each of the 25 wells. The well plate featured 5 different concentrations of cadmium, 10 nM, 20 nM, 30 nM, 40 nM, and 50 nM. These concentrations were chosen to fill the gap in data from previous characterizations. Each concentration was run with five wells run in parallel, additionally 5 wells of a control were run in parallel. The OD of each well was taken at the beginning of the experiment, then on the hour, every hour, for 5 hours reading both OD and florescence. The wells were excited using a wavelength of 532 nm, and detected at 588 nm. Final analysis preformed after Wiki Freeze.