How is plasmid transformation controlled?
Koen De Smet
k.desmet at nospam.ic.ac.uk
Tue Apr 28 02:30:15 EST 1998
Charles A Miller wrote:
> 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)
> oravaxcm at world.std.com
Plasmids tend to have "incompatibility regions" that prevent a second plasmid from
establishing itself in a bacterium that has a plasmid with the same region. But I don't
know if this mechanism is involved in preventing you from obtaining clones with two
plasmids after transfromation.
I think that during transformation, the odds are very small that you will get a single
clone acquiring two different plasmids. A typical transformation (or electroporation)
contains >10*8 bacteria. If they are very competent, you will still only get 10*5 or 10*6
colonies, so only 1/100 or less of the bugs have actually taken up a plasmid. So the
chance of getting a clone with two plasmids would be 1/10,000.
Koen De Smet
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Imperial College School of Medicine at St Mary's
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