I am trying to knockout gene in bacterial strain (genome sequence is available) so I must choose the first selective marker but I found that this strain is resistant to aminoglycoside, Blactum antibiotics as it contains contains genes such as Aminoglycoside phosphotransferase, multidrug efflux pump, Penicillin-binding protein.
I tried high concentration of antibiotic dose but it's still showing growth ... can anyone suggest what criteria I should choose or what to do now ?
Thank You Akshamal for information this paper is really helpful but I m not getting idea how resistance cassettes will be useful as my strain is resistant for most of the antibiotics please suggest or any article
Ok thanks for information now I am confirming is my strain is really resistant or not because its only insilico analysis prediction.
Try a bunch of different antibiotics and a bunch of different concentrations. Even antibiotics in the same family can have different levels of activity. For example I work with the thermophilic anaerobic bacterium Clostridium thermocellum. I've found that thiamphenicol is much more effective than chloramphenicol. Similarly, I've found that neomycin is more effective than kanamycin. It's difficult to determine antibiotic resistance from in-silico analysis. C. thermocellum has a gene annotated as an aminoglycoside phosphotransferase (which should inactivate both kanamycin and neomycin), but both antibiotics are effective at high enough concentrations. For kanamycin, I think the MIC (minimum inhibitory concentration) was 1500ug/ml, so make sure you go as high as possible with the concentration. Can you post a list of the antibiotics and concentrations that you've tried?
Thank you Daniel ... I tried Streptomycin, Kanamycin, Erythromycin, Chloramphenicol, Vancomycin, Penicillin, currently I tried only this antibiotics because of availability and all tried from 10 to 100 ug/ml
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