Part:BBa_K2969044
pCI434-TEVts6#-TCI42-pL(3)-pR(3)-mRFP
pCI434-TEVts6#-TCI42-pL(3)-pR(3)-mRFP contains the promoter of CI434 and TCI42, the coding sequence of TCI42 transcriptional repressor, and the reporter gene mRFP with RBSs and terminators. The promoter of CI434 contains the DNA binding site of CI434 transcription repressor. Once the CI434 binds to the promoter, the expression of genes under the promoter will be inhibited.
Characterization
We put the protease TEV protein under the promoter of CI434 to make CI434 as the inhibitor of the expression of TEV protease. Also, we insert the recognition site of TEV protease into the coding sequence of CI434 so that TEV protease can repress the function of CI434. When we combine the pCI434-TEVts composite part with the CI434-TEVsite expressing part, it will perform the function of cold-inducible switch.
TCI42 is the coding sequence of thermo-sensitive transcription repressor. Also, it is the cold-inactivated transcription repressor. We put the reporter gene mRFP under the promoter of TCI42 pR(3)-pL(3) so that heat can induce the expression of mRFP.
So with this composite part and expressing part of CI434-TEVsite, when the temperature is high, the sfGFP will be expressed. Otherwise, the expression of mRFP will be activated. So that the expression of sfGFP and mRFP can be controlled by the change of temperature. If we replace the reporter genes with the target genes, then our parts can be applied in more functions and therapies.
AS shown in Figure 1, our cold-inducible ON-switch works when the temperature is low and then express green fluorescence. When the temperature rises, the activity of cold-inducible ON-switch is inhibited and the heat-inducible ON-switch works. Therefore, we can turn on different genes’ expression at different temperatures. Similar with our single thermo-sensitive parts, this double-status switch also shows high performance and versatility.
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