For a detailed explanation of how Lambda Red recombineering works, check out our Lambda Red technology page.

For a step-by-step procedure, see Protocols.

Building the selection strain: Lambda Red Recombineering

Kan-ZFB-wp-His3-URA3

In this case, for the his3 ura3 system, we inserted, in order, a Kanamycin cassette, the zif268 binding site (ZFB), a weak promoter, His3, and URA3. The weak promoter 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.

Zeocin substituting rpoZ

In this case, the rpoZ gene is the bacterial homolog of the ω-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. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC209740/pdf/jbacter00169-0047.pdf D. R. Gentry and R. R. Burgess, Gene 48:33-40, 1986]

Fig.1) Gel image illustrating that zeocin has been inserted in place of rpoZ. Colonies 1 through 4 were picked off from a zeocin plate, and were thereby selected for zeocin expression. Lanes 1, 3, 5, 7 show the 650bp band that resulted from doing PCR with rpoZ_R (a primer flanking the rpoZ site) and zeocin_R (a primer internal to the zeocin cassette). Lane 9 has no band, as the control has the endogenous rpoZ and not zeocin at that locus, and zeocin_R can not bind anywhere. Lanes 2,4,6 show the larger band when using rpoZ_F and rpoZ_R (the two primers flanking the rpoZ region) to PCR out the zeocin cassette, compared to lane 10, where the same primers are used to PCR out the smaller rpoZ gene. Lane 8 shows no band as the PCR had not worked properly, but lane 7 shows that the zeocin cassette has been inserted into the bacteria from this colony.

Figure 2. Gel image illustrating that the Kan-ZFB-wp construct Kan-ZFB-wp construct has been inserted into the genome. Lanes 1,2,3,4 show the larger band as the Kan-ZFB-wp construct is about 1400bp in length. Lane 5, the control, shows a much smaller band as the endogenous promoter is only about 400bp long.