Difference between revisions of "Team:DTU-Denmark/Contribution"

The method used by the Cornell iGEM team in 2013 merely showed that the PtrpC promoter was able to provide GFP expression in a qualitative way. We wanted to examine how strong the promoter is and how much GFP it is able to express in a more quantitative manner.
The characterization of the PtrpC promoter was done by integrating the promoter into the plasmid, as described on the Design page. The promoter was characterized by two separate experiments. In the first experiment, the PtrpC strain was cultivated in quadruplicates in 50 mL breathable falcon tubes in 10 mL minimal media. A negative control consisting of the parental strain was included. The strains were cultivated for 80 hours at 37 °C with shaking at 150 rpm. The culture was then stored at 4 °C and subsequently, protein was extracted and fluorescence measured as described on the Experiments page. The resulting data is illustrated in figure 1.

Early GFP production

The trpC promoter was tested in seven different wells in the BioLector by measurements of fluorescence and biomass every 5 minutes in the 85 hours, during which the characterization experiment was running. The concentration of the GFP fluorescence units (GFP-FU) was calculated by a standard curve which was constructed from the results form the BioLector. The GFP-FU equivalent fluorescence is illustrated in figure 2.
From this figure, it can be seen that the TrpC promoter expresses GFP relatively early after inoculation.