Transconjugation problem - (May/28/2007 )

i'm currently stuck with my project in this process of conjugation. I'm using a suicide vector to construct a mutant to be delivered through E.coli S17 via conjugation. However, after many efforts and tries, i still couldn't get my mutant. Anyone have any suggestion or ideas for conjugation? Thanx in advance..

We often had trouble with conjugagtion too.

First I need a little info- from what strain are you mobile to and from? Also, do you need a helper plasmid with the mobilization functions (generally you do).

We often do tri-parental conjugation matings from E. coli into Pseudomonads. We found that if we mix the bacteria and place them onto a small filter, place the filter on plain TSA or LA, allow conjugation to occur overnight on the filter while the bacteria grow, and then select for the transconjugate of interest the next day this GREATLY increases our success.

Alternatively, depending on the strain you are trying to get the sucide plasmid into we found electroporiation to work VERY well! It works GREAT in most pseduomonas and all E. coli's I have tried. If you need a detailed protocol reply again and I will try to remember to check this website soon.

Cheers!

I'm using E.coli S17 carrying pSUP202 (clone with gene of interest) to conjugate with Burkholderia pseudomallei. I'm using filter membrane as well, place on agar overnight then wash and plated on LB + antibiotics. But it is hard to srceen for double cross over mutant. I heard of idea where colonies that grew overnight were picked and subculture into LB broth without any selection and incubated overnight at 22 degree without shaking to allow the recombination event.

Dear nielsen2,
Yes it would be great if you can get me the electroporation protocol because my plasmid is quite big around 10 kb. Also the journals that do electroporation on B. pseudomallei is low around 10^3. Thanks.