ecoli at cix.compulink.co.uk ("K N and P J Harris") writes: > > ==========
> > bionet/microbiology #6086, from chris.michiels at agr.kuleuven.ac.be,
> 1876 chars, Wed 04 Jun 1997 14:03:21 -0
> > Comment to 6078.
> > ----------
> > Article: 7138 of bionet.microbiology
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> > From: Chris Michiels <chris.michiels at agr.kuleuven.ac.be>
> > Newsgroups: bionet.microbiology
> > Subject: Re: Curing plasmids from a strain
> > Date: Wed, 04 Jun 1997 14:03:21 -0700
> > Organization: K.U.Leuven
> > Message-ID: <3395D819.7CB at agr.kuleuven.ac.be>
> > References: <916D87B9F at rose.le.ac.uk>
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> > Dr Martin Goldberg wrote:
> > >
> > > I have a strain with a pBR322-based plasmid and I want to cure the
> > > strain of this plasmid. Is there any way of achieving this without
> > > using agents such as ethidium bromide? I am considering
> competing-out
> > > the plasmid by transforming the strain with pBR322 or one of its
> > > other derivatives and selecting for this latter plasmid. Has anybody
> got an
> > > opinion on this approach or are there any more elegant methods?
> > > If your pBR322-derivative still confers tetracyclin resistance on
> the host (E. coli?),
> > you could try with a positive selection procedure for loss of
> tetracyclin resistance.
> > This has been described for E. coli, and is based on the fact that
> (pBR-based) Tc
> > resistance results in sensitivity to fusaric acid. Loss of TC
> resistance can be
> > selected by resistance to fusaric acid. This allows you to select for
> spontaneous loss
> > of the plasmid. If you don't find a reference, I'm sure I can find it.
> Somewhere...
> > Best regards,
> >
> > Chris Michiels
> > K.U.Leuven
> > Lab Food Microbiology
> > Leuven, Belgium
> A standard way of "accidentally" curing Rhizobium of plasmids is to keep
> them at elevated temperatures that are not "lethal". This is the least
> invasive way of losing plasmids.
> Peter Harris,
> University of Reading, UK.
>
You can also insert second plasmid of the same incompatability group as pBR322
with a different resistance marker you can select for. This is easy to do,
but of course you are left with new plasmid in your bacteria, which may or
may not be OK for what you are trying to do.
Adam
