restriction inhibition of transformation
malexeye at JAGUAR1.USOUTHAL.EDU
Wed Dec 3 20:41:38 EST 1997
Kevin Young wrote:
> I am attempting to move a plasmid from E. coli to an environmental
> isolate of Salmonella typhimurium. I can move the plasmid into the S.
> typhimurium strain TR5877, which is res(-)mod(+), and this is good enough
> to modify the plasmid so that it can be transformed into the normal
> laboratory S.t. LT2 strains. However, we can NOT get the plasmid into
> the environmental isolate, having tried all the regular transformation
> methods and electroporation. Thus, it seems to be a restriction problem.
> What is the simplest method for circumventing such a restriction problem?
I would bet on conjugation. Since you already have your favorite
plasmid in TR5877, you can use triparental mating using strain harboring
one of RP4 derivatives. Of course, you have to modify your plasmid by
inserting mob region. However, this shouldn't be a problem since
numerous mob cassettes available, some as small as 200-300 bp. Or you
can PCR up this region from RP4 that has been sequenced completely.
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