Dears,
Is there any one having any experience with knocking out tonB gene, to disrupt TonB dependent transporting system in bacterial?
we constructed the knockout plasmid but could not get any colonies after recombination. The knockout protocol works quite well before, and we suppose it would be some selection problem? The mutant will be sensitive to environment and can only grow with certain condition?
Much appreciate for your answer!
Cheers, Bin
You did not obtain colonies after recombination! I am thinking if you provide more facts -like have you established site(s) of your recombination event- I know the theory could be that your knock out plasmid crosses over at the tonB - have we ruled out secondary events ? Probably as you pointed out you may have got recombination and a knock out but your enviroment for selection may be hindering colonial growth . Probably you may have to fine tunes the selection environment in the presence of lack TonB transporting system
You shouldn't have a problem getting a tonB mutant in E. coli, those strains normally grow well enough. You can always do a tonB- selection using either phi80vir phage or one of the colicins (I think its ColB but can't remember for sure).
Thanks Michael, the strain I am working on is Pseudomonas putida, and I saw some papers sucessfully to get the mutants from E coli and also Pseudomonas aerugenosa, and that is why I am a bit confusing about the outcome. I will double check with the technician who did the work, and may just try once again. Cheers, Bin
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