In article <s.lee-0103960946190001 at sbs-43-144.sbs.auckland.ac.nz>,
s.lee at auckland.ac.nz (Soon A. Lee) wrote:
>> Dear net readers,
> I wonder if you could help me. My problem is this - I want to do northerns/growth curves on strains overexpressing certain genes from the gal1 promoter. The overexpression decreases the growth rate, meaning I can't harvest the strains all at once at log phase for analysis (if I grow from scatch on galactose). I would like to induce the promoter at say OD600 0.3-0.4, then allow four hours more growth for induction before harvest. What is the best way to do this? I have heard of growing on glycerol/lactate medium followed by galactose addition at the appropriate time (anyone have a description of glycerol/lactate synthetic medium?). However, for certain reasons I would prefer to use a fermentable substrate. Could I make medium with a low glucose conc, with normal galactose, and rely on depletion of the glucose for induction? How much glucose should I use to see depletion by about early log phase? Any tips would be much appreciated.
> Cheers,
> Colin MacDiarmid
> University of Auckland
> --
I would simply grow in glucose and then spin down and replace with
galactose. This would be the best way for getting reliable induction. I
worry that trying to be fancy about it by trying to get the exact
concentrations of galactose v.s. glucose right would required far more work
and many trial attempts to get it right. Also it is unlikely you will get
induction as good as switching media because you only need a small amount
of glucose to get pretty good repression. Glucose repression of the GAL1
gene takes a few hours to be relieved once you switch to galactose, but you
can monitor this easily using e.g. a GAL1-lacZ reporter or even performing
a galactokinase (GAL1 gene product) assay (e-mail me if you want details).
Don't try growing on glycerol/lactate because the growth will be quite slow
and if you are ultimately going to be overexpressing a foreign protein you
will find that protein synthesis will be relatively poor. Glucose -->
galactose is the best way to go.
-Marc Lamphier
University of Tokyo
