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In Vivo Characterization

Ptet Characterization

Since we are using the Ptet promoter in combination with TetR in our reporter systems, it was useful for us to characterize the Ptet promoter only. This characterization was done with the J61002 Ptet-RFP plasmid.

We added increasing concentrations of ATC in order to evaluate whether the DH5alpha cells that we used for the transformations expressed TetR; we also wanted to know the highest RFP expression we can get when Ptet is fully induced.

ATC induction

The DH5alpha cells can still exhibit a basal level of TetR expression, even if the plasmid we transform doesn't contain TetR. By adding ATC, we are able to inhibit this basal expression of TetR, revealing the full power of Ptet as a promoter sequence.

Cells without ATC:

Cells with ATC:

Experimental results:

There is a clear difference between cells untreated or treated with high doses of ATC. Still, this difference is not enormous, showing that our DH5alpha cells don't normally express much TetR. The basal expression of RFP is about 23'000 normalized RFUs, whereas with ATC we go up to 28'000 normalized RFUs.

Dose-response curve

The dose-response curve doesn't seem to saturate at the highest values of ATC added, indicating that some TetR proteins were perhaps still acting on the Ptet promoter. However, it is nice to see that TetR does have a quantifiable action on the promoter; it means that we can use it in our first readout system.