TOL plasmids

K.G. Korth kkorth at kcc.com
Tue Dec 12 11:33:50 EST 1995


rcaspi at sdcc3.ucsd.edu (Ron Caspi) wrote:
>Hi, is there a TOL plasmid expert out there?
>
>I am working with a strain of P. putida, and would like to check for
>the presence of such a plasmid. Does anybody have a protocol that will
>let me visualize such a plasmid on a gel?  (unfortunately, pulse field
>is not available).
>
>Many thanks in advance
>
>

I did my Masters thesis on TOL plasmid detection at SDSU (yes, right 
accross town). My thesis should be available at the SDSU library.  Since 
TOL is a low copy number plasmid, the Eckert (sp?) method of in-well 
lysis is grossly inadequate, since it doesn't give a strong enough signal 
to really make working with the plasmid possible.  If you want a TOL 
plasmid prep protocol use a modified Anderson and McKay (1983) Appl & Env 
Microbiol. 46:549-552. The prep was originally designed for isolating 
large plasmids out of lactic Streptococci, but it does wonders in 
isolating TOL plasmids. If you want TOL plasmid DNA sufficiently purified 
(no RNA) you will need to follow-up the plasmid prep with CsCl density 
centrifugation (CsCl centrifugation strait from a culture lysate will 
give you a pathetically low plasmid yield).  If you want the 
modifications, let me know [give me a fax #].  Its important to grow up 
the P.putida w/ TOL in a modified media to enrich for TOL plasmids as 
well (containing m-toluate and 4-methyl benzyl alcohol). TOL plasmid 
containg bacteria have a nasty habit of losing their plasmids (or having 
them transpose to the genome) if they are not being used.

Good luck.  TOL plasmids are easily one of the toughest plasmids to work 
with in Molecular Biology.  If you want PCR primers for the plasmid, I 
have some sequences that seem to work well [amplifying a region of the 
xylB upper pathway gene] that I never got around to publishing.  I am 
doing work TOTALLY unrelated to TOL plasmids since graduate school, so 
you are welcome to the (old) information.

Talk to Barbara Hemmingsen at SDSU.  She was my thesis advisor.  I'm not 
sure whether she has had students continue my work or not.

-- 
K.G.Korth
Kimberly-Clark Corp.
Neenah, WI 54956
E-mail: kkorth at kcc.com





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