plasmid curing

[Tom Anderson]

On Sun, 11 Mar 2007, Yoram Gerchman wrote:

> You will need a plasmid with the same origin of replication but another
> antibiotic resistance gene. Make competent cells from the bacteria you
> want to cure, transform them with the this extra plasmid and select for
> its resistance without the original plasmid resistance. Transfer on this
> antibiotic for a few times and then test to make sure you lost the
> original resistance (and hence the original plasmid).

I haven't heard of that method before.

The traditional method, as i understand it, is to treat the bugs with
acridine orange or ethidium bromide, which interferes with replication of
plasmids, but not the genophore. This is something that's been mentioned
on this list many times over the last decade and a bit - although i don't
think anyone's actually ever posted a protocol!

I believe the general approach with EtBr is to do a titration on normal
bugs (the most similar strain you can find) to make a kill curve, then
treat your plasmid-ridden bugs with the highest dose which doesn't cause
massive lethality. Continue for several doublings. Dilute, plate,
replica-plate onto selective medium, and pick colonies which are
susceptible to selection.

We have, however, had positive reports of the use of novobiocin:

http://www.bio.net/bionet/mm/methods/1995-March/026229.html

And coumermycin:

http://www.bio.net/bionet/mm/methods/1995-March/026198.html

Turning to the literature, apparently, psychotropic drugs can also be
used:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7008510&dopt=Abstract

And there's a general treatment of curing in:

Identification of plasmids at the genetic level - V. A. Stanisich (1988);
Meth Microbiol 21(Ch 2):17

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk