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