I'm trying to delete multiple genes from BL21(DE3) genome by P1 transduction.
My plan is using P1 transduction to transfer the deletions from Keio collection to BL21(DE3). Following some references, once getting a correct transductant, I have to remove the Kanamycin resistant gene flanked by 2 FRT sequences by expressing the FLP recombinase from a temperature sensitive plasmid called pCP20. That's clear to me.
However, I'm wondering what will happen after I introduce a second deletion and want to remove the Kanamycin resistant gene again. As I understand, there will be 3 FRT sites in the genome (one is the scar from the previous deletion and two are new). So, is it possible I will get some variations? Like, having deletions between any 2 FRT sites, including an unwanted region between two deletions?
And also, is there any step that I can optimize to get quicker deletion?
Currently I'm following the protocol described in Curr Protoc Mol Biol. 2007, E. coli genome manipulation by P1 transduction (I attached it below).
Thank you very much in advance