INVSc1，酿酒酵母酵母菌菌株

trp1-289突变是一种TRP-1基因的点突变，可导致该酵母株色氨酸营养缺陷（即，该酵母株的生长培养基中需要含有色氨酸）。许多载体含有TRP1基因的野生型拷贝，能够补偿trp1-289突变表型，从而可作为这些载体的筛选标记物。trp1-289突变与trp1完全缺失突变的表型相似，但是，与trp1-289不同，trp1完全缺失的酵母株不能被半乳糖有效诱导。因此，使用半乳糖诱导时，最好使用trp1-289突变株。但是，由于trp1-289突变株存在可检测频率的恢复，所以一定要对您的克隆进行验证。

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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