I have to perform transformation of a vector into clinical isolates of Klebsiella pneumoniae. My vector consists of chloramphenicol resistance gene but the isolates are resistant to chloramphenicol. Hence after transformation, i am able to get colonies even in the control plate (empty competent cell without vector on a chloramphenicol plate). How do i validate this.
I would recommend either removing the chloramphenicol resistance gene in the plasmid and replacing it with an antibiotic resistance cassette you know all of your strains are sensitive to, or use a similar plasmid that has a different antibiotic resistance cassette (if one is available).
You might try using a higher concentration of antibiotic. My lab used to work on Serratia marcescens and its was natively resistant to most antibiotics at the concentrations used for E. coli but was not resistant to higher concentrations. So for ampicillin we used 500ug/ml and for chloramphenicol or kanamycin we used either 100 or 200 ug/ml.
So if your strain just has background tolerance then increasing the concentration of antibiotic should be fine. However if your strain is truly resistant (and expressing CAT enzyme) them it will be resistant to the higher concentrations and you will need to change drug markers.
Varsha P Shetty that's a tricky one esp with MDR Klebsiella.
We have worked with NDM1 and CTX co-producing Kleb pneumo that was resistant to most of things.
With our strain, we screened weird eucariotic/procariotic active drugs, like hygromycin, geneticin, zeocin and hygromycin worked. So I would suggest to do susceptibility or MIC on these drugs and if there's some reasonable MIC, you can try to use that casette in your vector.
Also looking at higher concentration of chloramphenicol is a good start before going further.
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