Vector for tightly controlling expression of a cloned gene

Michael Benedik benedik at uh.edu
Fri Feb 21 11:52:21 EST 1997


In article <19C529A476B at rose.le.ac.uk>
mdg at leicester.ac.uk (Dr Martin Goldberg) writes:

> 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                   *
> *****************************************


We have had good luck with low copy number plasmids (pSC101, or pACYC
derivatives) carrying lac promoter and also carrying lacI gene to
ensure plenty of repressor is made. 

I haven't used it, but I have also heard lots of good stuff about using
arabinose promoter, it is tightly regulated.


Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at uh.edu



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