[putida]

Hey,
I have previously knocked out a gene of interest in pseudomonas putida, and replaced it with a gentamicin cassette. Surrounding the gent gene are FRT sites. This part seemed to work fine, the mutants grow on gent, while the wild type do not. However, now I am trying to electroporate a plasmid containing FLP into these mutants so that FLP can remove the gent resulting in unmarked mutants. On the FLP plasmid I have (pFLP2) is a beta-lactamase gene... however, when I electroporate into the mutants and select with carbenicillin, I get equal background growth (a full on smear) on the control plate (electroporated with no pFLP2) as on the pFLP2 electroporation plate. P. putida will grow on amp, and I found that my strain (KTT2440) contains a AmpC-type Beta lactamase gene (Class C Beta lactamase), as well as many efflux pumps to very inconveniently pump out antibiotics quickly before they can take effect. I have tried concentrations from 200ug/ml - 1mg/ml of carbenicillin, and although there will be less growth on plates at higher concentrations, the growth is still equal on the control plate as on the pFLP2 electroporated plate. I have also looked for a tetR FLP plasmid for use in P. putida, but have not found one in the literature... tetracycline was suggested as a much better and stronger antibiotic to use for selection in P. putida.

(I am making my comp cells from an OD600 of appx. 0.5 with 3 washes in 0.5M sucrose.)

Any ideas of a way around this would be incredibly helpful!