In article <5ekjs5$2p7 at Masala.CC.UH.EDU>, benedik at uh.edu (Michael Benedik)
wrote:
> > 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 *
> > *****************************************
>>> 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
The AraC regulated promoters found on the pBAD plasmids are excellent. In
the absence of arabinose and in the presence of glucose, they are really!!
off. Jon Beckwith's lab developed a whole series of them and they are
quite useful.
Good Luck,
Matt Nilles
Dept of Microbiol. and Immunol.
Univ. of Kentucky
