Protocol for Titrating Gene Expression Levels in Budding Yeast.

STAR protocols (2020-10-29)
Yimiao Qu, Jun Jiang, Xiaojing Yang, Chao Tang
ABSTRACT

The biological phenotype is affected by the level of gene expression. Here, we provide a step-by-step protocol for precisely titrating and quantitatively observing the target gene expression level in budding yeast by manipulating its copy number in the genome. Using this method, we construct various strains with different gene copy numbers of the cell cycle inhibitor Whi5. This protocol enables stable and inherent control of gene expression at the expected level with fluorescent intensity as the quantitative readout. For complete details on the use and execution of this protocol, please refer to Qu et al. (2019).

MATERIALS
Product Number
Brand
Product Description

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L-Glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
D-(+)-Glucose, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
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Yeast Extract, for use in microbial growth medium
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LB Broth with agar (Lennox), Highly-referenced microbial growth powder medium with Agar, low salt, suitable for salt-sensitive E. coli culture.
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Yeast Nitrogen Base Without Amino Acids, Yeast classification medium used for selecting yeasts based on amino acid and carbohydrate requirements
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L-Arginine, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Lysine, ≥98% (TLC)
Sigma-Aldrich
L-Isoleucine, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Methionine, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 99.0-101.0%
Sigma-Aldrich
L-Tryptophan, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 99.0-101.0%
Sigma-Aldrich
L-Phenylalanine, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Threonine, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 99.0-101.0%
Sigma-Aldrich
L-Serine, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Tyrosine, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 99.0-101.0%
Sigma-Aldrich
Adenine, BioReagent, suitable for cell culture
Sigma-Aldrich
L-Aspartic acid, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
Uracil, suitable for cell culture, BioReagent
Sigma-Aldrich
Peptone from animal tissue, from meat, BioReagent, suitable for cell culture, suitable for plant cell culture