When you passage pTargetF transformants, are you passaging them with kanamycin at 30° to maintain pCas?

I guess also after you get plasmid integration you'd also need to keep arabinose induction for the lambda recombinases to get a 2nd cross over to remove the plasmid from the chromosome, since you knocked out recombinase A.

Alexandra Johnson Hey thanks for coming back to me^^ So I usually confirm the genomic integration by colony PCR, in parallel i inoculate the same colony used for that in LB kanamycin at 30°C and after the integration is confirmed i add IPTG to that culture (i also tried adding IPTG straight away). For the second answer, i confirmed the location of the genomic integration by amplifiying the genomic integration with a primer which binds in the genome and with a primer which binds in my protein of interest to be inserted. I than load it onto a gel, purify the band and send it for sequencing. By that i think i confirmed that only the editing fragment and not the whole plasmid was inserted into the genome. However, i appreciate your idea and will try it anyway. Cheers

I'm attaching a slightly edited figure from this paper:

Article Technological Advances in Bifidobacterial Molecular Genetics...

It's not E. coli but the mechanism is pretty much universal in bacteria. When you provide a recombination template for deletion/insertion on a plasmid, the way the desired mutation happens is through a cross over where the whole plasmid integrates at one of the homologous ends, then in some cells a second recombination happens where there's a (theoretically) 50-50 chance the desired chromosomal mutation is achieved or the wild-type allele is restored. This is is normally mediated by RecA itself, not sure how efficient it is with short homology based recombinases like lambda's. Colony PCR at only one of the ends can look correct while still having the whole plasmid integrated.

Usually the way I check for gene deletions/insertions/plasmid removal is by using 3 primer sets:

One set that amplifies across the whole region where the deletion/insertion is happening (in the case of gene replacement this only shows up on a gel if the inserted gene is a noticeably different size than the wt).

One set that amplifies something only on the plasmid.

One set that only amplifies the wt gene.

Also, for passaging I would recommend streaking for isolated colonies multiple times rather than broth passage, broth passage gives a greater possibility for a mixed population where a small number of bacteria that still retain the plasmid get carried over each time.

Final suggestion would be to forgo having the recombination template itself on pTargetF, and to transform it in as linear DNA, the other method suggested in the paper. Your competent cells for transforming the linear DNA would need to have both pTargetF-yourgRNA and pCas in them and be induced with arabinose both before and after transformation to protect the linear DNA from degradation. Then you would induce with IPTG (probably on the recovery plates). Luckily that's relatively easy. It might work better than the template on the plasmid.

Let me know if you figure it out!