I have been working on creating on knocking out the ThyA gene (an essential gene) in e. coli using lambda red but have only been successful with lab type strains (MG1655 and BW25113), but unsuccessful with more pathogenic/"wild" strains such as NGF-1, 042, and MP1 for some reason. I am not sure what is the reason I can very easily do so with the lab type strains but have never been able to do so with the other strains.
Is there any other methods that could work to knock out this gene in these strains?
Can you provide more details ? Which lambda red system are you using ? Can you delete other genes in non-K-12 strains ? Do you add abundant thymine during and after outgrowth to complement the auxotrophy induced by the thyA mutation ?
Nathan Fraikin . I have been using essentially the same protocol from Datsenko and Wanner ( https://www.pnas.org/content/97/12/6640 ). I have the strains transformed with pKD46, induce them with arabinose, make them electrocompetent, transform with my PCR product derived from pKD3, recover in SOC media supplemented with Thymine and plate overnight with more thymine supplements. I have not tried other genes in the MP1 or NGF1 personally, but the MP1 strain has been used successfully with lambda red by colleagues in the past at different university and 042 has also been used successfully numerous times by a close collaborator for other genes as well. The two K12 strains have always been successful with lambda red for the ThyA gene.
I am not sure what alternatives there are to knocking out this gene in the other strains? CRISPR?
This is weird. Another way to isolate thyA mutants would be to plate overnight cultures on trimethoprim, pick growing clones and sequence their thyA genes.
Nathan Fraikin Thanks that seems like an interesting idea. I was wondering if you knew of a good protocol from a paper? I am having trouble being able to convince my PI since he is afraid of off target effects or mutations affecting the strain and was wondering how robust and specific this technique is?
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