Knock-out of bacterial gene

Bill Alexander alexanderw at cber.cber.fda.gov
Wed Apr 17 15:01:37 EST 1996


In Article <Pine.SOL.3.91.960417184759.5547A-100000-100000-100000 at tin>,
"Jose M. Bautista" <jbautist at hgmp.mrc.ac.uk> wrote:
>On Tue, 16 Apr 1996, Paul N Hengen wrote:
>
>> 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. 
>> 
>> 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"}
>> 
>> --
>>
*******************************************************************************
>> * Paul N. Hengen, Ph.D.
>
>.
>
>Yes, Paul, it must to be for a specific strain which have the properties 
>that are useful for my experiments.
>
> 
>Anyway, thank both -Paul and Tom- for your help. I will try soon your 
>suggestions since I have already the gene + CAT gene. In preliminary 
>experiments I have observed that my cells can adquire Cm resistance. I 
>wonder if it will work for the selection. Could you suggest any other 
>selection system (No Tet since my strain is already knocked-out in 
>another gene with Tet).
>
>Thanks again
>
>Jose
>
Try Sac B system.  You can select for insertions and then against the
plasmid to knock out your gene.  

Donnenberg, M. S. and Kaper, J. B. Construction of an eae Deletion Mutant of
Enteropathogenic Esherichia coli by using a positive-selection suicide
vector. Infec. Immun. 1991, 59, 4310
Regards,

Bill Alexander
alexanderw at cber.cber.fda.gov

     "640K ought to be enough for anybody." -- Bill Gates, 1981



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