triparental conjgation prblm

Rafa Maldonado Rafael at genetics.med.utah.edu
Tue Jan 25 15:51:21 EST 1994


Hi Shahram
I have some experience in triparental matings in gram-negative
bacteria, but I have no idea about cyanobacteria, so maybe I'm saying
many nonsenses.
I understand that your shuttle vector is a tra+ plasmid, and your
cosmid a mob+ cosmid. If not, you cannot do this mating. Also the tra
capacity of your plasmid should work in trans. Here are my advices:
1.- I have found that triparental mating is very ineficient, and it
would be advoided if you can put your tra+ plasmid in the same cell
that your cosmid is. They have to have diferent replication origins.
You can change the library to a coli with your shuttle vector and you
may get an improvement of the frequency of transconjugants of 100 fold
or more.
2.- Other amazing possibility thay you can try: put the plasmid in the
Anabaena (because you say it is a shuttle plasmid, I undertand that it
can be replicated in cyanobacteria) and do the mating with the library.
It works in some bacteria and plasmids.
3.- Also, I have found that very long incubations of the mating plate
don't improve the frequency, but it drops several magnitudfe orders.
Try reducing the incubation time; but I think it'll depend on the
biology of Anabaena, and  I don't know if it possible do that.
4.- Increase the amount of cells in the mating, mainly the donors
(both). Concentrate x10 the amount of cells in the mating. Also, some
bacteria don't like conjugation, so perhaps you have to concentrate the
Anabaena only. Try!

Good luck!

----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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