Gene knockout in E.coli

Mikhail Alexeyev malexe at biost1.thi.tmc.edu
Wed Aug 14 08:27:03 EST 1996


In article <4uscnn$cq6 at mserv1.dl.ac.uk>, pra1 at essex.ac.uk wrote:

> In article <morby-1408960934540001 at mnemosyne2.bioc.cf.ac.uk>,
morby at cf.ac.uk (Andy Morby) writes:
> |> Has anyone seen a recent paper on an E.coli strain which is permissive for
> |> gene knockout by homologous recombination i.e by introduction of a neo
> |> cassette etc.  I'm sure it exists but can't seem to source it by the usual
> |> channels.
> |> 
> |>       Thanks
> |>                Andy Morby
> 
> I understand this can be done using a recD strain. This allows
> transformation with linear DNA containing your gene with the neo
> cassette insert, or some other selectable marker. The recD gene 
> encodes a subunit of the exonuclease that would otherwise degrade
> any linear DNA before homologous recombination had a chance to 
> take place. I have not tried this method myself.
> 
> Peter Alefounder.




It can also be done in recBC, sbcB (C) strains like ATCC 47002. Works fine
with Cm-res marker. Km-res (Tn5) is somewhat worse.

M.A.



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