I have bacterial strains (E. coli) that are drug-multiresistant and pathogenic. I would like to delete some genes in those strains using the "One step inactivation of chromosomal genes" (Datsenko and Wanner, 2000). However, I can hardly select my mutants because my transformants strains are resistants or intermediary resistant to almost all antibiotics.
Does anyone knows an alternative selection for my mutants (without antibiotics) or an alternative protocol for gene deletion?
Why they use in Denmark with Aspergillus fungy is to use heterotrophic strains and they never need any antibiotics. Much cleaner way. And they can remove the selection gene after it is not required anymore. Of course creation of such heterotrophic strain would be difficult at first but once you’ve got it, it would accelerate your progress.
Well, the strain is heterotrophic for some amino acid or vitamine or something. And you use the missing gene instead of antibiotic resistance gene, so that your transformants are autotrophic (i.e. grow on media without the supplement contrary to the WT).
And they even used system where they could remove the selection gene easily. The strain was uracil or thymine dependent and they used some gene recovering this. Thus after transformation it was able to grow without U/T (I’m not sure which one). However, if they added 5-fluoro-uracil, it produced some toxic product and thus only fungi lacking the selection gene survived.