I have used Kanamycin resistance as a selection marker with kanr-containing pET vectors.
Since KanR is inactivating the antibiotic (only in the cytoplasm), I was wondering about the duration of kanamycin selection pressure. So far, I haven't faced any problems and if any, the easy solution is to re-add kanamycin to the medium, but how long is a standard selection (say 50ug/ml) effective?
Thank you in advance for your answers,
Dear Dr Singh,
thank you for your answer.
Indeed, Kanamycin itself is not extremely stable, and when a colony grows on a plate, the thickness of the overgrown colony can allow the "highest" bacteria to loose contact with the antibiotic. This can (as I heard) be either due to the inactivation by the resistance marker, or also by diffusion problems.
Myself, in order to keep my transformed bacteria, I didn't face problems yet with storing them for a week at 4°c. However, it was only with E.coli. I have no real experience with other bacteria
Do you have some experience with planctonic cultures? Is it needed to re-add kanamycin for more than 24 hours long cultures at 37°
Dear Bhoj,
Yes, indeed. but Kn is also inactivated by KanR, thus the concentration of active antibiotic decreases over time. after a certain time (which I don't know), the Kn concentration drops below the MIC of susceptible E.coli. this time is much longer than for Ampicillin, but non nil, and I wanted to know an estimate.
also, my worries were for protein overexpression : if after induction, a bacteria looses the plasmid, thanks to this active-Kn depletion, it will overgrow the transformed bacteriae and the protein yield will drop.
if I only express the protein over a few hours, it doesn't cause any problem, but a colleague of mine wanted to do an overnight expression, at 37c (which isn't usually advised) and It thus raised this question. (It is also important to say the protein accumulates as inclusion bodies, to be further refolded, so causes toxicity issues and may act as negative selection markers)
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