Gene disruption

Michael Benedik bchs1b at Elroy.UH.EDU
Thu Mar 19 13:18:10 EST 1992


In article <1992Mar18.175031.13419 at urz.unibas.ch>, herzer at urz.unibas.ch writes:
>
>I am attempting to disrupt a gene in a strain of E. coli which is not well
>characterized (the strain, not the gene).  I have a plasmid construct which
>carries my gene with a chloramphenicol resistance gene inserted into it.
>So if I get a double crossover of homologous recombination then my strain,
>which is Cm-sensitive, should then become Cm-resistant.  I am using a
>temperature-sensitive mutant of pGB2 which grows at 30C but is kicked out
>at 42C.  The problem is that there are a high amount of revertants with
>this plasmid and I have a very high background.  Does anyone know of any
>other good plasmids which could be used for this purpose; i.e. temperature
>sensitive or "suicide" plasmids,etc.  I would be most greatful for any 
>suggestions.
>
> Thanks,
>                        Pete
>                        "Herzer at urz.unibas.ch"

A lot of people use lambda phage to do gene replacement.
There are a lot of plasmids that do what you want. A recent reference to
one is Gene vol 105 pg 17-22, This plasmid requires IPTG to replicate
and stops replication in the absence of IPTG. The suggest that it will
do gene replacement and integrative suppression. Let me know if you
need other references or help designing the experiments. Personally
I have always done this with lambda, but then I like lambda.
With the plasmid you are using, is there another marker on the 
plasmid? Could you just screen by replica plating or toothpicking throuhg
all the colonies which arise at 43C for loss of function of your gene
or for loss of another plasmid coded resistance among those which are
cam R at 43. Although it won't be 100% you might find what you want
say at 1% so you just need to toothpick a few hundred colonies. This
would be much faster than getting a new vector and subcloning.
	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU



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