Team:DTU-Denmark/Contribution

Characterization

The Cornell iGEM team from 2013 showed that the Aspergillus nidulans promoter PtrpC is able to express GFP in Cochliobolus heterostrophus by using fluorescence microscopy. We wanted to further characterize this part.

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.

Figure shows fluorescence/protein for negative control and Trpc. Negative is quantitatively smaller.Fig. 1: In the second experiment, the expression of GFP over time from TrpC was measured using a microbioreactor (Biolector, m2p-labs). class=""/>

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.

Graph showing development in GFP expression which increases over the first 40 hours, then decreases for 10 hours and then stabilises.Fig. 2: The Figure shows the expression of GFP-FU where it enters the experimental stage after 5 hours and peaks after 40 hours with an approximate concentration of 14 nM. The expression stabilizes at approximately 10 nM. class=""/>
In the figure 3, it can be seen how much GFP is expressed per biomass. It shows that a considerably high amount of GFP is expressed, compared to the amount of biomass in the beginning of the BioLector run. This can be explained by the fact that the biomass had not yet had time to grow, and did not enter the exponential phase until the BioLector had run for about 10 hours. After 10 hours, a decline in the expressed GFP per biomass can be observed. This is to be expected, as the biomass grows with a considerably higher rate than than the expression GFP.
he Figure shows the expression of GFP-FU per biomass. It increases for the first ten hours to approximately 0.14 nM GFP-FU/biomass. It declines afterwards and is stabilized at a concentration of around 0.05 nM GFP-FU/biomassFig. 3: The figure illustrates the expression of GFP-FU per biomass during the 85 hour BioLector run. class=""/>


In conclusion, the trpC promoter expresses GFP, as measured by fluorescence as well as fluorescence per biomass. This has been measured quantitatively and we have seen that the expression of GFP is 14 nM GFP-FU on average at the highest points of expression. Furthermore, we have seen that the promoter behaves consistently between biological duplicates until 55 hours of growth.



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