Dear friends

I am trying to construct few deletion mutants of Mycobacterium abscessus using the pJV53 recombineering system. I have transformed the M. abscessus cells harboring the pJV53 plasmid (carries recombinase, Km resistant) with the linear PCR product which contains the flanking regions of the gene to be deleted and the zeocin cassette. I selected the colonies in 7H10 plates containing kanamycin and zeocin. However, when I do PCR to confirm the incorporation of zeocin cassette into the gene to be deleted, I get the same band as that of wild-type. I checked the resistance of M. abscessus WT cells to kanamycin and zeocin and the cells didn’t grow in both the antibiotics.

Any help on this would be greatly appreciated.

Warm regards

Bindu