Gene deletion

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    • #17511
      clefg
      Participant

      Hi!

      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?

      Thanks for your help!

    • #114308
      JackBean
      Participant

      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.

    • #114310
      JackBean
      Participant

      BTW this multi-drug resistency is achieved how? Is it on plasmid or chromosome?

    • #114312
      clefg
      Participant

      Thanks for your relpy! Maybe I wasn’t clear enought but I want to delete genes in my drug-multiresistants strains without changing the rest of the bacterial strain…

      The drug resistancy is naturally found in both plasmid and chromosome.

      Anyway, I am curious to know how does it works with the heterotrophic strains?

    • #114313
      JackBean
      Participant

      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.

    • #114376
      Cat
      Participant

      Use the same protocol, just modify it – substitute Luciferase or other fluorescent protein gene in place of an antibiotic resistance gene.

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