E.coli "gene knockout": help

Robert Solomon Bioc rgs at mole.bio.cam.ac.uk
Mon Mar 6 08:17:13 EST 1995


borsang at dibit.hsr.it (Giuseppe Borsani) writes:

>Does anybody have any suggestion on how to create a "gene knockout" in the
>E.coli genome when no selectable phenotype is available for the gene to
>inactivate?
>Probably it's something obvious for people with experience in bacterial
>genetics.  References and protocols are welcome.

References for gene knockouts in coli..

Gutterson, NI & Koshland, DE Jr, Proc. Natl. Acad. Sci. USA 80, 4894 - 8 (1983)

Gay NJ, J. Bacteriol. 158, 820 - 5 (1984)

Basically, these involve a temperature sensitive polA mutant strain, wherein
the plasmid carrying the mutated gene cannot replicate extrachromosomally at
the restrictive temperature - thus, growth on antibiotic at restrictive temp
indicates a recombination event (hopefully a homologous recombination event).
This leaves you with two chromosomal copies of the gene, separated by the
plasmid DNA.  Selection of segregants (loss of plasmid ) should leave you
with a mix of wt and mutant bugs.  How you select between them is up to
you - if you have no screen for function then I suspect it's up to PCR
and sequencing to rescue you - I'm having no joy PCR'ing from either
colony scrapings or chromosomal DNA preps, but I'm assured that these are
possible.  P1 transduction is then used to take the mutation into the
background strain of your choice.  Have fun.

Good luck

Rob Solomon



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