Knock-out of bacterial gene

Jose M. Bautista jbautist at hgmp.mrc.ac.uk
Wed Apr 17 12:59:01 EST 1996


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

.................................................................
.       Jose M. Bautista                                        .
.       Department of Haematology                               .
.       Royal Postgraduate Medical School                       .
.       Hammersmith Hospital, Du Cane Road, London W12 0NN,UK   .
.       Tel + 44 181 743 2030 ext. 2162                         .
.       Fax + 44 181 742 9335                                   .
.       e-mail boxes:   bauchem at eucmax.sim.ucm.es               .
.                       jbautist at hgmp.mrc.ac.uk                 .
.                       jmbautista at samba.cnb.uam.es             .
.................................................................




More information about the Methods mailing list