In article <glarson-191295121010 at 126.96.36.199>, 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
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
Dept of Biochemistry and Molecular Biology