INVSc1 S. cerevisiae Yeast Strain

The trp1-289 mutation is a point mutation in the TRP-1 gene that causes this strain to be auxotrophic for tryptophan (i.e., tryptophan is required in growth media for this strain). There are many vectors that contain a wild-type copy of the TRP1 gene that will complement the trp1-289 mutant phenotype and can therefore be used as a selectable marker for such vectors. The phenotype of a trp1-289 mutant and a trp1 complete deletion mutant are similar, but strains with the trp1 complete deletion, unlike trp1-289, will not induce well with galactose. Therefore, when galactose induction is used, it is better to use a trp1-289 mutant. However, since trp1-289 mutants revert with a detectable frequency, it is important to verify your clones.

S. cerevisiae can grow using either or both mechanisms of carbon metabolism. The balance between the two is different for glucose vs. galactose as a carbon source. Under ideal conditions, S. cerevisiae grows slower on galactose than on glucose, because production of glucose-6-P from galactose is rate limiting. (gal -> gal-1-P -> glu-1-P -> glu-6-P). Under non-ideal conditions (low oxygen, as in the center of a colony or a culture without really good oxygen feed), it becomes even worse because cells grown on galactose are using more respiration than fermentation relative to cells grown on glucose. Low oxygen makes fermentation more necessary, which cells growing on galactose are not good at.

As with other sugars (e.g. glucose), D-raffinose is the biologically active carbon source for yeast. Pure L-raffinose will not work.

The suggested initial cell density for galactose induction is 1 to 5 X 10E6 cells/ml . The cells are allowed to divide one or two times and then induced with galactose. Galactose induction is best in log phase and the culture will probably approach static phase at 1 to 4 X 10E7 cells/ml. Induction of cells maintained in raffinose may begin in 15 to 30 minutes whereas induction of cells maintained in glucose may not first occur for an hour or more. Peak expression will often occur in 2 - 4 hours so time points should be taken every hour (or every other hour) for up to 10 hours. When using raffinose maintained cells, the induction is much faster than induction of glucose maintained cells. Maximal expression levels remain the same.

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The following are commonly employed auxotrophic markers:

1) his3Δ1: Histidine requiring strain (from gene disruption) with a deletion in locus 1. The his3 denotes the disruption of the HIS3 gene. The Δ1 is a deletion that has been engineered to decrease the recombination between the incoming plasmid DNA and the chromosomal site.
2) leu2: Leucine requiring strain due to the disruption of the LEU2 gene.
3) trp1-289: Tryptophan requiring strain, developed from gene disruption and a further point mutation to decrease the recombination between the incoming plasmid DNA and the chromosomal site.
4) ura3-52: Uracil requiring.

For more detail on types and methods of gene disruption in yeast refer to METHODS IN ENZYMOLOGY Vol. 194.