Currently, my project involves the preparation of Pseudomonas aeruginosa mutants and one of the steps involves triparental mating to transform P. aeruginosa with the mutant plasmid.
My question is: why not transform the P. aeruginosa with the plasmid through heat shock or electroporation?
without knowing the experiment you wish to do, it is not possible to give you an accurate answer. But the likely explanations could be that one or more of the plasmids are too large to easily purify and transform with good efficiency. It could also be that you are needing a large population of various transconjugants and the number is higher than you might get through transformation.
Ah I apologise for not being clear.
My goal is to mutate certain proteins in P. aeruginosa so I've constructed plasmids which are to be eventually transformed into P. aeruginosa to facilitate mutation through homologous recombination of the insert from the plasmid into the genome.
Ergo, I've first transformed donor E. coli cells with the plasmid construct and these cells will then go through triparental mating with PAO1 cells and RK600 helper E. coli cells.
I suppose what you said makes sense. I appreciate your help. Thank you so much!
My guess is that it is a numbers game, Pseudomonas transformation is not always that efficient and with mating you can generate and screen very large populations.
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