In article <4iou15$a7m at mserv1.dl.ac.uk>, vachon at bio.grenet.fr (Gilles
VACHON) wrote:
> Hi all,
>
>What I have to ask to this newsgroup is probably kind of silly but here
it goes.
>I am not a microbiologist but for some reasons (I don't want to go into
>details but will if you want me to) I need to make an E.coli strain with
a
>modified functional lacZ gene (that I have almost made already) inserted
in
>its genome.
>snip<
Hello, Gilles
Take a look at this paper: de Lorenzo, V. Herrero, M. Jakubzik, U. and K.
Timmis. 1990. Mini-Tn5 Transposon Derivatives for Insertion Mutagenesis,
Promoter Probing, and Chromosomal Insertion of Cloned DNA in Gram-Negative
Eubacteria. J. Bacteriol. 172:6568-6572.
The short version is that you can clone your fragment of choice into a
plasmid carrying mini-Tn5, you can then deliver that plasmid via
conjugation into your recipient strain (the E. coli strain you want your
lacZ gene to go into). The transposon will randomly insert itself into the
recipient genome. You can then select a few independent hops to make sure
the phenotype that you are seeing isn't due to some insertional
strangeness.
The paper has the address for Dr. Timmis.
Good Luck,
Brad
===========================================================================
Brad Nicholson |"If it worked the first time,
Department of Pathology | it wouldn't be research."
University of Utah | ...Brad Nicholson
Salt Lake City, UT 84132 | My opinions are solely my own.
Brad_Nicholson at hlthsci.med.utah.edu |
or: (801)-581-4365 | iligitimi non corborundrum
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