Hi,
I have to do a bacterial conjugation experiment for the first time. My donor strains are fosfomycin resistant isolates of klebsiella pneumoniae and the acceptor is E. coli J53. However I have no colonies on the conjugation plates after the experement. I hope there is just a problem in my procedure somewhere. Here it is:
Any and all advice would be greatly appreciated!
Thank you
You need some to add some additional information for a complete answer. What conjugation system are you using? is the fosfomycin resistance plasmid encoded and is the promoter functional in E.coli? have you titrated the selection level to your recipient strain? is the plasmid stable in E.coli?. You might want to grow the overnight of the donor under selection since if the plasmid is not stable it will be lost during overnight growth. It is true that some plasmid are inhibited for conjugation during stationary phase, therefore it is good you are growing to log phase, However you can simplify the experiment by directly using stationary phase cultures (just add 0.5ml of each to an eppie and let them sit for an hour at 37C then plate (overnight conjugation might be excessive depending on the plasmid). Finally some conjugation system preferentially work much better in solid phase mating rather than in liquid (aprrox 1000 fold difference). Try a solid phase mating (take a dense inoculum of cells from an agar plates of each donor and recipient and streak them on top of each other on a fresh LB plate then allow them to sit at 37C for 3 hours following by using a loop to pick cells from the cross-streaked position (you wont see an actual colonies just collect with a loop from the trace on the agar) to the selection plate.
Thank you for your suggestion. However this is the first time for me to do conjugation experiment. I am sorry that I am not very familiar with the detail of the experiment. So I just followed some article which focus on fosfomycin resistant klebsiella pneumoniae or E. coli as the articles (e.g. DOI: 10.1089/mdr.2018.0137). .
I have read the article. If you look at the result section it says they used filter mating (although it says broth mating in the materials and methods) this indicates they were successful with solid mating therefore you should do that. Also I suggest you go with MH and not LB since that what they were using.
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