Hi,
I have a doubt about the cloning of antibiotic resistance genes. how do I choose the antibiotics to add to the medium when I need to select the recombinant clones? I know that one antibiotic should be that carried by the vector, but what about the other antibiotic that allows me to see which clones bring the resistance genes?For example..I am working on a strain that is resistant to a given carbapenem. I don't know exactly what mechanisms trigger the resistance, even if I suspect they include resistance genes. When I digest the DNA of this bacterium and I transform e.coli with cloning vectors carrying each a DNA fragment, how do I choose the most suitable antibiotic to detect the recombinant clone having the resistance gene? I am in doubt if to use carbapenems for the selection, because they are quite powerful and in my strain the resistance to this antibiotic could be the result of different mechanisms, not only the expression of a given gene..
I hope to have been clear.
Thanks
You should be able to directly select for carbapenem resistance assuming that the mechanism of resistance is truly a resistance gene. Be sure that your vector uses an entirely different antibiotic marker for vector manipulation (for example you should probably use a vector encoding resistance to Kanamycin, or chloramphenicol or tetra cycline). You can select for recombinant clones using the vector resistance to be sure you have built a good library and then select subsequently for carbapenem resistance.
On the other hand, if the mode of resistance in your strain is through a different tolerance mechanism then it may not function in as a dominance resistance marker in trans on a plasmid.
Agreed with Michael, one thing I would suggest if you are worried about the use of carbapenem then plate onto media with lower concentrations of that antibiotic. That may let you see some more subtle effects.
And remember to always select for the vector that you have as well!
Hi Silvia, you should use in the same plate both carbapenem (to select strain) and the antibiotic to wihch the vector confers resistance (to select recombinant strain).
You can do a minimal inhibitory concentration (MIC) assay using your original strain to determine the baseline antibiotic resistance level for the antibiotics of your choice. You can then use the MIC value for the selection of your transformed bacteria to identify the colonies expressing the particular antibiotic resistance gene.
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