Knock-out of bacterial gene

Paul N Hengen pnh at cutterjohn.ncifcrf.gov
Mon Apr 15 16:23:58 EST 1996


Jose M. Bautista (jbautist at hgmp.mrc.ac.uk) wrote:

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

If you already have the gene cloned, it would be easy. Here are some ideas
in decreasing efficiencies...

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.

2. Make synthetic linkers to place on the ends of an omega with enough homology
to cause an insertion by crossing over like above.

3. Use a transposon which has insertion specificity or resides in the
gene while on a plasmid in trans. This would work best using a suicidal
entry system.

--
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