How is plasmid transformation controlled?

Charles A Miller oravaxcm at world.std.com
Tue Apr 28 00:19:19 EST 1998


I'm sure this is a basic question, but I just can't seem to find the
correct search terms in Medline. My basic question is this: 
How do gram-negative bacteria conrol the number of plasmids
that are taken up during transformation (natural, electroporation..).
How does E. coli do this? I am curious since it seems that when one
makes a plasmid library in E.coli, you can isolate single plasmids
(not a mix of plasmid) from a given colony. Do other gram-negative
bacteria usually behave this way? Or is it a specific mechanism? If it is
a mechanism, is there a way to control it or introduce it into
other gram-negatives? If I want to try to introduce a unique gene into
one isolate of a bacteria by creating a random library of DNA (from
an isolate that DOES have the unknown gene) in a plasmid shuttle
vector, then screening the transformants for the phenotype, how
can I be sure that each bug is only getting one plasmid and not
several? I suppose that one could then narrow it down to the one 
plasmid if more than one are taken up, but I was wondering what the 
"rules" are for gram-negs?

Please keep in mind that this is just a rough little demo which probably
wouldn't work ll that well, but I'm really looking for mechanism
by which E. coli (and others?) accepts a single plasmid and no other.
I hope someone can help me out with this question or point out some
references (since Medline wans't too helpful)

Thanks,

Chuck
oravaxcm at world.std.com




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