Team:NTNU Trondheim/Alternative systems

From 2011.igem.org

Alternative system

From the results, promoter rrnB P1 is not transcribing enough repressor to inhibit pLac-promoter. To test out if the other parts of the system work (LacI-pLac-mCherry), a test system where Pm-promoter controls expression of LacI was made. The wild-type Pm-promoter that was used here is induced by m-toluate.

To be able to transfer the system from pSB1A2 to expression vector pVB20wt they were both cut with XbaI + PstI. The system was inserted into pUC128.

LacI+LacP+MC in plasmid pUC128 was isolated.To retrieve the LacI-pLac-mCherry insert pUC128 was cut with EcoRI and the fragments were separated on gel. The insert fragment were cut out and extracted from the gel.

Figure 1: The Puc128 plasmid with LacI-LacP-MC cut with EcoRI. The square indicates the LacI-LacP-MC insert

To transfer it to pVB20wt, pVB20wt was opened using EcoRI and then treated with CIP to avoid religation, and was then ligated to the EcoRI cut insert fragment.