I am planning on conducting P. aeruginosa conjugation using E. coli S17-1 lambda pir as donor strain.
I have also read some publications and studies that were using Pseudomonas Isolation Agar (PIA) to select against E. coli. But here instead i am going to use Simmons Citrate Agar as the media to screen the transconjugants against the E. coli. It is based on the fact that E. coli's growth is inhibited when cultured on SCA, and P. aeruginosa is not. Has anyone ever experienced using SCA in P. aeruginosa conjugation?
and is it possible to use SCA with antibiotic for better selection (such as 100 ng/ul gentamicin)?
We did the conjugations in LB overnight, and used LB plus streptomycin to confer-select E. coli.
Thanks for the reply Mr. Cornelis, I tried using chloramphenicol 30 ug/ml and it was very selective against E. coli cell.
Hi Samuel, did you get to conjugate the P. aeruginosa using that method? I am having problems to transfer a suicide plasmid (pEX18) from E. coli S17 to pseudomonas sp (isolated by my advisor). Do you have some protocol that you may share it?
Hi Eric Velasco
Yes, I got my mutant using this technique. I couldn't really remember the details as it was a long time ago when I did this experiment. I remember referring to the protocol below and slightly adjusted the details
https://openwetware.org/wiki/Dandekar_%26_Chandler:Pseudomonas_Mating
As I mentioned in previous answer, I used LB + 30 ug/ml chloramphenicol (instead of PIA or SCA) to select against E.coli
I also used NoSalt LB + Sucrose (as explained in the protocol) to select my Pseudomonas mutants against merodiploids, but wasn't really successful, so I check the mutation using PCR and got 2 mutants out of 40ish merodiploids.
Good luck.
Dear Samuel Widodo and Eric Velasco
The following chapter describes a conjugation method that can be really useful for you!
Engineering Gram-Negative Microbial Cell Factories Using Transposon Vectors
https://pubmed.ncbi.nlm.nih.gov/27709582/
Best,
Samuel Widodo Thanks for share the protocol and your experience about the sucrose counter-selection. It seems a very labourious method since the amount of mutants in contrast to merodiploids produced. Well, I hope to have a bit of lucky.
Edmar Ramos Oliveira-Filho Thank you so much! I am going to read your mentioned chapter. Indeed, I think it will be very useful for us.
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