I have a rather complex problem. I am trying to delete a regulatory
gene in a pathogenic strain of E. coli. I have managed to achieve this
in a K-12 strain, no problem. However, it appears that deleting this
gene in the pathogenic strain is lethal. So far, I have introduced a
suicide plasmid containing the deletion with which I want to replace
the wild-type allele, into the strain, to form a merodiploid. However,
when the plasmid recombines out of the chromosome, normally, 50% of
the resolved merodiploids should be wild-type, the remaining 50%
should be mutant. However, I only get the wild-type allele back. I now
have plent of evidence to indicate why deletion of this gene should be
lethal.
What I therefore propose to do, is to clone the wild-type gene minus
its own promoter into a plasmid containing a tightly controlable
promoter (lacZ is too leaky). I will then transform the merodiploid
with this construct and incubate in the presence of low concentrations
of inducer, to permit low levels of expression of the plasmid copy of
the gene. Hopefully, when the suicide plasmid recombines out of the
chromosome, the chromosomal copy of the gene will be replaced by the
mutant allele. Thus, with only the controlable copy of the gene on the
plasmid, I can study the expression of virulence genes regulated by
this gene. I am not interested in trying to overexpress the gene (VERY
LETHAL!!!!!), simply to control its expression. Please can anybody
help?
*****************************************
* Dr Martin Goldberg, *
* Dept. of Microbiology and Immunology, *
* University of Leicester, *
* University Road, *
* LEICESTER. *
* LE1 9HN *
* UK *
* Tel. +44 (0)116-252 3017 *
* Fax. +44 (0)116-252 5030 *
* E-mail mdg at le.ac.uk *
*****************************************
