I am working on generating a mutant of Acinetobacter baumannii ATCC 17978 using the pEX18 suicide plasmid to create a knockout (KO) of the ompW3 gene.
During the first selection step with gentamicin, I can successfully isolate colonies that carry the plasmid. However, in the final selection step with sucrose, where the plasmid should be eliminated and only the KO mutant bacteria should remain, I observe the growth of wild-type (WT) strains instead.
Does anyone have any ideas about what might be happening?
Are both regions of homology (on each side of the disruption) of similar size? You should in theory get equal numbers of both if they are.
Also, are you positive that your suicide plasmid only has a single copy of the insert?
Lastly, are you working with multiple independent insertion events or did you just pick one? Sometimes just using a different colony might help.
Hello Michael J. Benedik , here are my answers to your questions:
Thank you very much for your help.
One thought is that you may be getting carryover from your original plates of wt. You might take 4 or 6 of your original 20 and restreak them out for single colonies on gentamicin a second time and then select on sucrose after you have truly pure stocks.
It might also be that a KO of ompW3 is either lethal or at a strong selective disadvantage so the wild type will outgrow it. Especially given your comment that the second selection everything grew much faster.
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