To target specific genes we worked with the CRISPR-Cas9 system. By designing the sgRNA, we can target different genes. At first, we targeted the RFP with Cas9 by electroporating our plasmid.
Figure 1: [left] control producing RFP, [right] Transformed strain
For the transfer of genes, we worked with conjugative plasmids (pSW23T and pK18mob).
We tried at first, a conjugation without killing. We transferred our conjugative plasmid pK18mob containing the LacZ gene coding for the beta-galactosidase. So The receiver strain, that produces RFP, will then acquire this gene and will appear purple on Xgal media from producing RFP (red) and metabolizing Xgal (blue).
Figure 2: [Red] Receiver strain, [Blue] Donor strain, [purple] Conjugated Receiver strain
We know from now on that our conjugation is working. We can now combine the two tools.
Thus, our next step is to target the RFP gene with our conjugative plasmid containing Cas9.
Cross test 1: [left] overview, [center] close up conjugative donor strain with sgRNA targetting RFP (+), [left] close up conjugative donor strain without sgRNA targetting RFP(-).
We can see that the conjugation line is visible under the microscope and an effective difference between the control, without sgRNA, and the conjugative strain with the sgRNA.
The last step aim at targetting a resistance gene in the receiver strain.
Conjugation in plate test 1: [left] : Donor and recipient strain after conjugation. [right] : control and recipient strain after conjugation.
Want to know more about our experiments? See our page Results