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
=====================================================================
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
