Hi,
I try to make Rifampicin resistance microbes (e.g., Corynebacterium) using point mutation in RpoB gene.
The doubling time of Corynebacterium is ~3.5hrs, and spontanoeus mutation rate for the average gene is 0.000000001.
Is there any protocol or method which induce point mutation in RpoB gene and gain Rifampicin resistance w/o or w/ mutagen?
Thanks in advance.
In the likely absence of sophisticated tools for the genetic engineering of your Corynebacterium, your best bet may be to use old-fashioned random mutagenesis (UV, nitrosoguanidine etc.) and to select for rifampicin-resistant mutants. You may try several cycles of selection with increasing concentrations of antibiotic. After obtaining a set of resistant mutants, you could identify those mutated in rpoB by PCR + sequencing the rpoB gene.
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