I am trying to make a knockout mutation of Campylobacter jejuni. I already in the final step and all I need to to transform my vector with the flanking regions of my gene and the antibiotic resistant marker to CJ strain 11168-o. However, in previous attempts I learned that the restriction-methylation system of CJ will cut destroy any attempts of transforming an E. coli plasmid to CJ. A technique that has been suggested by the Gaynor lab was to use extremely high amounts of DNA during the transformation (~20ug). I did the experiment and after 4 days of incubation I finally have one tiny colony growing in my plate.
Could this colony be my knockout mutant?
The answer to this question will be in our next episode!