Hi all,

We are trying to recombine a WT gene with mutated gene in Klebsiella pneumoniae chromosome. We are using a recombineering method described by Li et al (ref: https://doi.org/10.1093/nar/gkt1075) that uses tetA-sacB positive-negative selection. The media used for counter selection is LB-20% sucrose with fusaric acid and ZnCl2 . The problem however is that following electroporation and recovery/outgrowth for 6 hrs to overnight the negative control cells (comps cells + NO DNA) too grow in the counter selection media. It is not just few spontaneously mutated colonies, a ton of colonies grow in the plate! We have tried both 37C and 42C but both didn't help. We tried streaking negative control cells shortly after electroporation and it is totally inhibited by counter-selection. So, our best guess is that whatever is the issue is happening during recovery/outgrowth phase.

So I was wondering if anyone having experience with tetA-sacB recombineering have any suggestion to have an effective counter selection?

Thanks!