Hi,
I have to do conjugation experiments to see if the resistance is transferred by plasmids. I don't have a strain in lab to be used as acceptor for my conjugation experiments, so I have to buy one. It is the first time I do this experiment. I was wondering, how do I choose this acceptor strain? Where do I get it? How do I select one suitable for the conjugation? I am reading plenty of papers where the authors say to use certain species of strains, but I can't understand where they find them, where to get them, how they decide the strains are ok for their conjugation.. Can you explain me?
Thank you.
Hello Silvia, you should start to answer this question:
Does the resistance is transferred by plasmid from one species to another (from K. Pneumoniae to E. Coli for example)
Or
Does the resistance is transferred from one strain to another (from a resistant K. Pneumoniae strain to a sensible K. Pneumoniae strain)
I think you can purchase any reference strain of the species of your interest.
I usually perform conjugation from E. Coli to P. Aeruginosa so if you need any help I'll be glad to help you.
Hi Silvia,
The strains used as recipients in conjugation experiments are usually classical lab strains with one or two chromosomal antibiotic resistance markers introduced by point mutations (rifampicin, novobiocin, streptomycin, fusidic acid...). This is because when you select transconjugants you need to select both for the (putative) transferable marker and one or two markers peculiar of the recipient strain only.
In principle you could build yourself a recipient by taking one strain, make sure it is sensitive to all the antibiotics, render it resistant to one of the antibiotics above and use it, even if it would be advisable to have the genome sequenced.
If needed, I can give you advice on some gram positive reference strains used as conjugation recipients.
Anyway, which species are you working with? Which kind of genetic element do you expect? Which resistance marker are you investigating?
Hope that helped,
Francesco
Thank you for your answers.
I am working on resistance genes of Klebsiella and I thought to perform the conjugation from a resistant strain (that is what I have in lab) to a sensitive strain of Klebsiella. Technically, I am also interested in seeing if the resistance is transferred to other species, like E.coli. If you can give me more suggestions in terms of what strains to choose, I would be very grateful..Also,how can I know if my donor strain will be also resistant to the antibiotic for which the acceptor strain chosen will be resistant?
Thanks
Where you get the strains is highly dependent, you could get them from ATCC, or from a stock center, or from your lab's strain collection or from some other lab in the vicinity. The type of strain you need as a recipient is one which can be selected for when mixed with the donor. So for example if your donor strain had an auxotrophic marker (His-) and your recipient strain was His+ then you could select on minimal medium without Histidine. Or you could have your recipient strain carry a drug resistance marker that the donor does not have. Frequently in E. coli they use a streptomycin resistant recipient in conjugation experiments, especially because spontaneous mutations to streptomycin resistance are quite rare. You could also just get a strain you wish to use from your collection and select for a resistant variant, using for example rifampicin or nalidixic acid which are probably not markers you are going to be looking at.
As to how you know whether your donor strain will be resistant, you just test it on the selective medium.
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