In article <glarson-191295121010 at 18.104.22.168>, 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?
No, you don't have to use mob vectors (BTW, in the case of vectors mob is
not a gene but rather a small DNA region, as small as 112 bp, that confers
to the vector the ability to be conjugally transferred). There are some
nice vectors out there (see Kaniga et al., Gene (1991) 109:137-141, for
example). Unfortunately, in some cases you have to make them work (at
least it was so in my case).
You can use the "yeast" approach in E. coli. Unfortunately, it works only
in strains with particular genotype: recBC, sbcB (sbcBC); or recD i.e. in
strans deficient for ATP-dependent exonuclease V (sbcB mutants lack
exonucleaseI), but still proficient in recombination (recD mutants have
elevated recombination proficiency). All other strains would "chew up"
your linear DNA. You may use "yeast" approach on such strain (like JC7623,
available from ATCC) and then transduce your mutation with P1.
Alternatively, if you use pMB1 (ColE1) replicon-based plasmid (with intact
rop gene, i.e. intermediate-copy-number vector like pBR322), you don't
even have to linearize it. This approach has been described by P. Balbas
et al. in Gene (1993-1994). The method is based on the fact that in recBC
mutants replication of ColE1-based plasmids is abberant. They form long
linear concatemers that are poorly segregated without selection. I tried
that approach, works nice. Yet another approach has been described in the
article published in 1989 in J. Bacteriology. Last author is S. Kushner
(Kushnir?). They used termosensitive for replication derivative of pSC101.
At higher temperatures it integrates into the chromosome. When you shift
temp. down afterwards the vector is being excized from the chromosome
carrying either wt or mutant copy of the gene. Again, there is no need to
linearise plasmid or perform transduction. However, it didn't wor as nice
as published when I tried it.
Sorry for not providing more detailed reference: I don't have them handy.