Lambda Red

Lambda red recombineering makes use of homologous recombination systems to allow the insertion of constructs into the genome. It is accomplished in two steps, as shown in the diagram and the procedure below.

Figure 1. Lambda Red, PCR to get the required insertion product (zeocin in this example) 915px

Applications of Lambda Red recombineering

Kan-ZFB-wp

In this case, for the his3 ura3 system, we inserted, in order, a Kanamycin cassette, the zif268 binding site (ZFB-Zinc Finger Binding site), and the weak promoter that has low levels of transcription on its own, but high levels of transcription when bound to the omega subunit that is attached to the ZFP (Zinc Finger Protein). After recombineering, the bacteria were plated on kanamycin agar plates to select for the insert.

Tet-ZFB-wp

After we got the selection system working with the zif268 protein and binding site, we swapped out the ZFB for the other sequences, and switched out the Kanamycin cassette for a Tetracycline cassette. This allowed us to change the binding site and select for cells that had the changed binding site, and the new ZFB

zeocin substituting rpoZ

In this case, the rpoZ gene is the bacterial homolog of the omega subunit on the expression plasmid for the ZFP (Zinc Finger Protein). In order to bind the level of transcription of his3-ura3 to the expression of the ZFP, it would need to be knocked out, so that there would be a reduced level of constitutive expression of the his3-ura3. Since rpoZ is a RNA polymerase subunit, knocking it out would reduce the viability of the bacteria, so we could not simply knock it out using MAGE. As a result, we used a zeocin cassette to confer an antibiotic resistance to the bacteria, which we then selected for through zeocin agar plates.