Knock-out of bacterial gene

Paul N Hengen pnh at cutterjohn.ncifcrf.gov
Tue Apr 16 18:22:52 EST 1996


Tom Bickle (bickle at ubaclu.unibas.ch) wrote:

| Does anyone have any good protocols/ideas for knocking-out a bacterial 
| gene (E.coli) of known sequence?

> 1. Insert a small omega fragment with an antibiotic resistance gene into the
> middle of the gene you want to knock out and transform the linear DNA into E.
> coli.  Select for resistant clones.

: This is not going to work too well in a wild type host because the recBCD
: enzyme is hell on linear DNA. you need a strain where the recBCD enzyme is
: inactivated and one of the alternative recombination psathways is
: activated. See, for example:
:
: Eddy, S.R. & Gold, L. (1992). Artificial mobile DNA element constructed
: from the EcoRI endonuclease gene.   Proc. Natl. Acad. Sci. USA 89,
: 1544-1547.

You are right. Although I didn't quite explain the whole thing above, and it
wasn't really clear from the post if this had to be a specific strain, I was
thinking of a selection system like that described in this paper:

@article{Winans1985,
author = "S. C. Winans
     and S. J. Elledge
     and J. H. Krueger
     and G. C. Walker",
title = "Site-directed insertion and deletion mutagenesis
with cloned fragments in {{\em Escherichia coli}}",
journal = "J. Bacteriol.",
volume = "161",
pages = "1219-1221",
year = "1985"}

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