Hi everyone!
I would like to insert a plasmid (containing genes for resistance to Gentamycin and Chloramphenicol) into an Erwinia amylovora strain resistant to Rifampicin.
So far, I've tried three different methods: electroporation, biparental mating, triparental mating, none of them succeded.
Does anyone have any experience in Ea transformation? Is it so difficult?
Thanks,
Anna
Have you checked your plasmid for restriction sites for which that species has restriction enzymes?
Do you know that this plasmid will replicate in Erwinia? Perhaps that is the problem.
It is a common pBBR plasmid and many people used it. So I think that the plasmid is not the problem
Do you know which pBBR plasmid you have, one of the common ones (pBBR122) I believe is NOT capable of being conjugated so bi and tri- parental matings will not work. Other ones might be. But certainly electroporation should work.
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