Info. needed on "suicide" gene for coli

Brad Nicholson nospam at nospam.edu
Wed Jan 13 10:05:21 EST 1999

In article <369CB6B9.509599B5 at bms.com>, "Pramathesh S. Patel"
<patelp at bms.com> wrote:

> Hi,
>         I am interested in getting info. regarding any gene I could use
> as a "suicide" gene in E. coli (i.e.. expression of a particular gene
> causes cell death).  I have tried the mammalian GATA-1 gene and the 
> Bacillus sacB gene.  Unfortunately, I have had no success.
>         Any help will be appreciated.  Thanks in advance.
> Prem
> patelp at bms.com

Hi Prem,

I have used the sacB system in gene knockout/replacement experiments in
Salmonella.  How did you try the sucrose counter-selection?  Several
papers have reported that there shouldn't be any NaCl in the medium and I
know that the amount of sucrose may have to be increased or decreased
depending on which background you are using.  Growing the bacteria at
lower temperatures for a shorter time (30 C for 16 hrs.) seems to reduce
the background.

A couple of other things come to mind.  First, you could try tet
(tetracycline resistance gene).  This allows a positive selection, Tet
resistance, as well as a negative selection, chlortetracycline. 
Alternatively you could use a WT rpsL allele (sensitive to Streptomycin)
in a rpsL mutant background (Strep. resistant). Other ideas that are not
conditional, ccdB, used in the Invitrogen pZero plasmid and any of the
plasmid "killer" systems like sok/hok.

If you are interested, I can give you some references to start with. Let
me know if I can be of specific help. You might consider trying to
optimize the sacB system by eliminating salt in the plates and checking a
dose response with the sucrose.  

Best of luck,

Brad Nicholson                      |"We've left Occam's Razor and  
Department of Pathology             | now are proceeding to Occam's 
University of Utah                  | Trash Can."
Salt Lake City, UT 84132            | Schooler/Burke 11-98
Brad_Nicholson at hlthsci.med.utah.edu | My opinions are solely my own. 
or: (801)-581-4901                  | iligitimi non corborundrum

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