Biparental mating (conjugation) and transformation are two entirely different things. Which one did you mean because the answers would be very different.

Thanks Michael J. ,Benedik for your feedback, I meant the biparental mating to introduce the gfp marker.

Doing a bi-parental mating is very simple, essentially you just mix the strains together for a while and then plate on selective media. For maximal efficiency you can take 50ul of each culture in early stationary phase, spot them one on top of the other on an LB or similar plate, and let sit for 8 hours or overnight. Then scrape up the patch of cells with a sterile loop, resuspend in broth or saline and plate out on selective media plates.

The tricky part is to be sure that your strains are correct, your donor strain needs to have the plasmid you wish to transfer but also the conjugative apparatus on another plasmid or integrated in the chromosome. And you need to have selection against each of the parent strains but for the correct ex-conjugant strain.

If you provided more information then I or someone else here can help you troubleshoot the experiment.

Thanks Michael J. Benedik , I appreciate your help, I almost did the some protocol that you suggested, after putting the mix of bacteria in selective media (100 ug/mL ampicillin) what I observed that my wild type strain, as control, was able to grow in this selective media. I am going to increase the concentration of the antibiotic, and I will see.

what are the relevant genotypes and the Selection and counter selections that you are using?

I am using minimum M9 media, as I understood in other protocols, E coli doesn't support growing in this media, while my pseudomonas dose.

It depends upon your strain of E.coli, but most strains designed for biparental mating do carry auxotrophic mutations so they don't grow in minimal M9. However for Pseudomonas most labs use carbenicillin instead of ampicillin, it seems to work better for Pseudomonas. And you may need to go much higher, such as 500ug/ml or even 1000ug/ml for either drug.

Michael J. Benedik I really appreciate your advices, thanks for your time, I will increase the concentration, about the use of carbenicillin, in my case the plasmid contain ampicillin marker.