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Titering Gal1 promoter

Kevin Morano kamorano at ucdavis.edu
Wed Aug 30 12:53:23 EST 1995


In article <jjschm-300895094600 at jjschm.monsanto.com>,
jjschm at ccmail.monsanto.com (Jon Schmuke) wrote:

> Hi all,
> 
> Does anyone know the best way to titer expression from the Gal1 promoter?
> I am trying to get a handle on the expression level needed for an essential
> enzyme that we knocked out in S. cerevisiae and replaced in pYES vector
> behind the GAl1 promoter.  Is it better to down regulate it by induction
> with galactose or by repressing with glucose in the presence of high
> galactose?  Or is there a better expression system for controlled
> expression?
> 
> Any help or comments appreciated.
> 
> Jon
> 
> ***************************************************************
> Jon J. Schmuke                       jjschm at ccmail.monsanto.com
> **************************************************************

Hi Jon,

   I used this vector for protein depletion, and found it to repress very
well.  It did not seem to overproduce massively, which was fortunate for
me. We grew cells in gal/raf and shifted to glucose to shut off.  I think
it is still pretty much off with a gal/glc combo but I don't think I've
ever tried it.  See what your expression is normally.  The other option is
to subclone the whole thing into a CEN vector, since pYES2 is 2u and
already establishes a rather high level.  Good luck!

Kevin Morano
kamorano at ucdavis.edu



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