Gene replacement in E. coli

[Philip Heacock]

In article <glarson-191295121010 at 151.112.27.59>, glarson at ccmail.llu.edu
(Garry P. Larson) wrote:

> I would like to test the phenotype of coli strain bearing a mutation in a
> non-essential chromosomal gene.  Therefore, what's the simplist way to do a
> knock-out gene replacement in E. coli?  Do I have to use some vectors with
> the Mob gene or (if so, could you contact me so I may use it)À
> Alternatively, can I  utilize an approach taken from my yeast experiences?
> Can I just take my cloned "gene-de-jour", place a Kan (or equivalent)
> cassette into it, then transform my strain with a linear fragment
> containing the resistance cassette flanked by a reasonable (200-500bp+) of
> homology on either side of the cassette and finally  select for drug
> resistance?  I've essentially done a similar  approach (w/o the drug
> cassette) in yeast to mutate a gene on a plasmid.  Seems like it should
> work or am I dreaming?
> 
> Any refs for the techniques would also be greatly appreciated.
> 
> Thank you
> GLarson 

Garry,
Try the method referenced in, The journal of Bacteriology, vol 175 #13
pages 4260-62. The title is "Simple Phagmid-Based System for Generating
Allele Replacement in Eschericia coli" by Steven Slater and Russell
Maurer.

-Phil Heacock

UTHSC-Houston
Medical School
Dept of Biochemistry and Molecular Biology