We are trying to use a gentamicin resistance cassette as a selectable marker in E. coli and Klebsiella. The cassette was PCR amplified from the CRIM plasmid pAH153 described originally by Haldimann and Wanner, and subcloned in another plasmid. The problem is that when we grow strains carrying pAH153 and the derivative mentioned above in gentamicin (10 ug/ml), cell growth is very variable, sometimes ranging from no growth or poor growth, to normal growth. Trying to find an explanation for such variation I found in the literature some evidence indicating that gentamicin activity is highly dependent on pH of the culture medium. So, the questions are if any of you have experimented similar issues with gentamicin selection in gram-negative bacteria, and if you use a medium with adjusted pH for doing so. Thanks
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