E.coli "gene knockout": help

Chris Yost cyost at acs.ucalgary.ca
Sat Mar 11 14:40:23 EST 1995

> >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. 
> Yes, PCR does work, and is the best (only?) way to go if you don't have a
> phenotypic/enzymatic assay (even if you do have an assay, it is still the way
> to go!)  (genomic DNA preps from selected colonies)

> > 
> > Good luck
> Seconded.
> > 
> > Rob Solomon
> Richard Heath

There is a way to directly select for gene replacement.  In other words
directly select for a double recombination event (ie no wt bugs).  The
strategy is described in Quandt and Hynes, 1993.  Gene 127:15-21. 
Basically it involves placement of the sacB gene in your suicide deliver
vector.  In the presence of sucrose, expression of the sacB gene is lethal
in gram negative bacteria.  Therefore after you have placed you suicide
vector construct in your bug, selection with your antibiotic resistance
marker on media containing 5% sucrose should yield bugs that have
undergone a double recombinational event (ie they did not incorporate sacB
into their genome).  To verify proper insertional inactivation of your
gene a Southern blot using your gene as a probe should show an increase in
size corresponding to the insertion of the antibiotic resistance gene.

I hope my description is clear and that this info helps. 

Chris Yost
cyost at acs.ucalgary.ca

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