I am making a deletion of a gene (size -400bp) in the genome of a gram negative bacteria Xanthomonas campestris pv. vesicatoria by triparental conjugation method. While screening the mutant strain by PCR from both ends of flanking region of gene I am getting two bands in mutant strain, one band is of correct size as expected for the mutant strain and second band is just above but in the wild type strain there is only one band as expected.What could be the reason for appearing a second band in the mutant strain ?
How did you make your construct? Need more detail but I guess you might got merodiploid. Your whole deletion construct was integrated into the chromosome. That why you still got your wild-type band. Your gene haven't deleted yet.
Hello and thanks for the reply. I cloned 800-900 bp of up and downstream sequence (of the gene to be deleted) in a cloning vector and then these sequences were ligated together and cloned in a suicidal vector. Suicidal vector containing ligated sequences of up and downstream to the gene was transformed into DH5 alpha pir strain. Further, triparental conjugation was performed between XCV WT strain, helper strain and DH5alpha pir strain and allowed to conjugate O/N. Afterward, triparental conjugate was plated and screened by different combination of antibiotics in an appropriate media.
From your information, I think they are merodiploid which can be resolved after second recombination occur. When the second recombination happen, you will get either mutant or your wild-type back. you can read this paper and apply with your project.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765137/
Hope this help
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