How to insert a lacZ gene into E.coli genome ?

Brad Nicholson Brad_Nicholson at hlthsci.med.utah.edu
Sat Mar 23 11:52:43 EST 1996

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 
 >modified functional lacZ gene (that I have almost made already) inserted 
 >its genome.

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 

The paper has the address for Dr. Timmis. 

Good Luck, 

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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