Description

This composite produces constitutively the CymR regulator protein, that binds the Cumate Operator repressing the transcription from the promoter.

Assembly

Obtained by synthesis.

Results

CymR expression was confirmed by Western Blot analysis. Our CymR has a SV5 tag at the C-terminus in order to be detected by an anti-SV5 Ab. Its functionality was confirmed through the repression of T5 Cumate Operator-GFP.

The validation of the repression of the protein CymR on the T5 cumate operator was verified through the creation of a plasmid containing a single copy of CYM R and the T5 cumate operator upstream the GFP (I13504). We saw that the expression of the GFP was completely repressed in absence of p-cumate but it was active in presence of p-cumate.

Moreover we saw that the integration of the double copy of CYM R is possible.

Modelling

Looking forward

As we test that a single copy of CYM R in the plasmid can repress strictly the T5 cumate operator the next step is two integrate in the genome of the bacteria first a single copy of CYM R then the double copy. If this integration is verified we can have together with the T5 operator and the toxin a simple and efficient system for the control of bacteria proliferation and for the elimination of the horizontal transfer.
Therefore we will clone a double copy of the CYMR in the genome and in the plasmid the T5 operator-Holin-T5 operator-LL37. If horizontal transfer occurs the bacterium that receives the plasmid is going to die because of the lack of repressor.

Link to the Registry