Wild‐type Saccharomyces cerevisiae cells (BY4742, MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0; ThermoFisher Scientific, Slangerup, Denmark), pda1Δ (BY4742, pda1::KanMX; ThermoFisher Scientific), and cit1Δ (BY4742, cit1::KanMX; Open Biosystems) were cultured in synthetic complete media (US Biological, Salem, MA, USA) supplemented with 12C614N2‐lysine (SILAC “light”) or 13C615N2‐lysine (SILAC “heavy”). Cells were harvested at the indicated time points, washed once with sterile H2O, and resuspended in lysis buffer (50 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA, 1x mini complete protease inhibitor cocktail (Roche, Basel, Switzerland), 5 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM beta‐glycerophosphate, 10 mM nicotinamide, and 5 μM tricostatin A) at ~50 OD600 cells/ml lysis buffer. The cell suspension was frozen drop‐wise in liquid nitrogen and ground in a liquid nitrogen chilled steel container by the Retsch MM 400 Ball Mill (Retsch, Haan, Germany) for 5 min at 25 Hz. The lysate was thawed, NP‐40 and sodium deoxycholate were added to a final concentration of 1 and 0.1%, respectively, and clarified by centrifugation. The lysate supernatent was precipitated with four volumes −20°C acetone. The acetone precipitate was dissolved in urea solution (6 M urea, 2 M thio‐urea, 10 mM Hepes pH8.0) and protein concentration determined by Quick‐Start Bradford assay (Bio‐Rad, Copenhagen, Denmark).
By4742
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
The BY4742 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a standard laboratory yeast strain widely used in scientific research. The product's core function is to serve as a reference strain for genetic and molecular biology studies.
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Lab products found in correlation
Complete mini protease inhibitor cocktail, by Roche (2 mentions)
Quick start bradford assay, by Bio-Rad (2 mentions)
D16 10, by Thermo Fisher Scientific (1 mentions)
Bp1422 2, by Thermo Fisher Scientific (1 mentions)
Bp1420 2, by Thermo Fisher Scientific (1 mentions)
By4741, by Thermo Fisher Scientific (1 mentions)
Bl21 de3, by Merck Group (1 mentions)
Round bottom glass tubes, by Thermo Fisher Scientific (1 mentions)
10 protocols using by4742
1
SILAC Yeast Proteome Extraction
2
Yeast Strains for Phenotypic Analysis
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The yeast strains used for phenotypic analysis in this study were the wild-type S. cerevisiae strain BY4742 (MATα his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0) (Open Biosystems, Huntsville, AL, USA), S. boulardii Sb.P (van der Aa and Jespersen 2003 (link)), Enterol S. boulardii—a strain isolated from food supplement (CNCM I-745 commercial Enterol® strain, Biocodex, Warszawa, Poland), and evolutionary engineered strains of CNCM I-745 Enterol S. boulardii ev16 and ev17 (KMUTT, Bangkok, Thailand). The yeast strains were routinely grown in yeast extract-peptone-dextrose (YPD) medium containing 1% yeast extract, 2% Bacto peptone, and 2% dextrose. Meanwhile, the evolution experiment was performed in YPD with an increasing concentration of acetic acid, adjusted to pH 4.5 with HCl or 4 M KOH.
3
Cultivation of S. cerevisiae Deletion Mutants
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S. cerevisiae strain NRRL Y-12632 (Agricultural Research Service Culture Collection, Peoria, IL, USA) was maintained and cultured on a synthetic complete medium. Nonessential haploid S. cerevisiae deletion mutations generated by the Saccharomyces Genome Deletion Project and the parental strain BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) were obtained from Open Biosystems (Huntsville, AL). BY4742 is a designed deletion strain directly derived from Saccharomyces cerevisiae S288C, which is widely selected as parent strains for the international systematic Saccharomyces cerevisiae gene disruption project48 (link).
Freshly grown cells harvested at logarithmic growth phase were used as inoculate after 16 h incubation with agitation of 250 rpm at 30°C. Cells were incubated on SC medium using a flask fermentation system at 30°C as previously described15 (link).
Freshly grown cells harvested at logarithmic growth phase were used as inoculate after 16 h incubation with agitation of 250 rpm at 30°C. Cells were incubated on SC medium using a flask fermentation system at 30°C as previously described15 (link).
4
Yeast Growth in Glucose Media
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The wild-type strain Saccharomyces cerevisiae BY4742 (MATa his3D1 leu2D0 lys2D0 ura3D0) from Open Biosystems/Dharmacon (a part of GE Healthcare) was grown in YP medium (1% yeast extract, 2% peptone; both from Fisher Scientific; #BP1422-2 and #BP1420-2, respectively) initially containing 0.2% or 2% glucose (#D16-10; Fisher Scientific) as carbon source. Cells were cultured at 30°C with rotational shaking at 200 rpm in Erlenmeyer flasks at a ″flask volume/medium volume″ ratio of 5:1.
5
Yeast Stress Response Evaluation
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The parental wild-type strain BY4742 (MATα his3Δ1 ura3Δ0 leu2Δ0 lys2Δ0) and its isogenic knockout mutants (gcn2Δ and hsp104Δ) were purchased from Open Biosystems. The double knockout mutant fes1Δhsp104Δ, which is also isogenic to BY4742, was established as previously described (10 (link)). Yeast cells were cultured in synthetic defined (SD) medium (2% glucose, 0.67% yeast nitrogen base w/o amino acids, 20 mg/L uracil, 30 mg/L L-lysine HCl, 100 mg/L L-leucine, and 20 mg/L L-histidine HCl) with reciprocal shaking (120 rpm) at 28 °C, and exponentially growing cells were harvested at OD600 of 0.5 to 0.6. Stress treatment procedures have been previously described (10 (link)). To determine cell proliferation activity, cells subjected to severe ethanol stress in SD medium were diluted, plated on YPD plates (2% glucose, 1% yeast extract, 2% peptone, and 2% agar), and incubated at 28 °C for 48 h. The number of colonies formed on YPD plates was counted to calculate CFU. Propidium iodide (PI) staining was performed using the method described by Davey and Hexley (79 (link)).
6
Yeast Cultivation for Optical Density
7
Propagation and Validation of Yeast Strains
8
Screening for DNA Suppressors of NEDD4w4
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E. coli strain DH5 was used for plasmid propagation and BL21(DE3) (Millipore) for protein production. The yeast Saccharomyces cerevisiae strains used in this study are: INV (Invitrogen), PJ69-4A (James et al., 1996) , MHY501 (Chen et al., 1993) , YYM4 (Stawiecka-Mirota et al., 2008) , BY4741, BY4742, BY4741 atg2 BY4741 atg13 , BY4741 atg14 , BY4741 atg18 (Open Biosystems), BY4741 LAS17-GFP (Invitrogen), CRY2040 HSP104-yTagRFP-T and CRY2041 HSP42-yTagRFP-T (Malcova et al., 2016) .
Yeast media used were YPD (1% yeast extract, 1% peptone, 2% glucose), SC synthetic complete medium (0.67% yeast nitrogen base without amino acids, 2% glucose) with desired supplements (adenine, uracil, amino acids (aa)); SCgal contains 2% galactose, and SCraf contains 2% raffinose instead of glucose.
To compare the growth of strains, middle-log phase cultures were diluted with water to have 1 OD 600 equivalents/ml. Aliquots of five-fold serial dilutions of these cell suspensions were spotted onto the medium and incubated as indicated.
Multicopy suppressor screen for DNA fragments restoring growth of yeast cells bearing pYES-NEDD4w4 (with URA3 marker) (Gajewska et al., 2003) was performed using a yeast genomic library from ura3 strain on the YEp351 multicopy plasmid (gift of M. Wysocka-Kapcinska, IBB PAS).
Yeast media used were YPD (1% yeast extract, 1% peptone, 2% glucose), SC synthetic complete medium (0.67% yeast nitrogen base without amino acids, 2% glucose) with desired supplements (adenine, uracil, amino acids (aa)); SCgal contains 2% galactose, and SCraf contains 2% raffinose instead of glucose.
To compare the growth of strains, middle-log phase cultures were diluted with water to have 1 OD 600 equivalents/ml. Aliquots of five-fold serial dilutions of these cell suspensions were spotted onto the medium and incubated as indicated.
Multicopy suppressor screen for DNA fragments restoring growth of yeast cells bearing pYES-NEDD4w4 (with URA3 marker) (Gajewska et al., 2003) was performed using a yeast genomic library from ura3 strain on the YEp351 multicopy plasmid (gift of M. Wysocka-Kapcinska, IBB PAS).
9
Yeast Desiccation and Stationary Phase
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The yeast Saccharomyces cerevisiae wild type strain BY4742 (Matα his3Δ leu2Δ met15Δ ura3Δ) and its derivative HSP26-GFP were purchased from ThermoFisher (Waltham, MA). Cells were grown in 15 ml round-bottom glass tubes (Fisher Scientific, Waltham, MA) to log- (14 hrs) or stationary phase (three days) in YPD medium (1% yeast extract, 2% peptone and 2% dextrose) at 30°C with constant shaking at 250 rpm. Cells were centrifuged (× 1,000 g for five min) and the culture medium was discarded. The culture tubes without caps were placed in a humid chamber (23°C, 50% relative humility) and cells were allowed to desiccate for 14 days [20 (link)].
10
Yeast Genetic Engineering Protocols
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The yeast strains and plasmids used in this study are listed in Table 1 . S. cerevisiae BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0; purchased from Thermo Scientific, Pittsburgh, PA, USA) was used as the yeast host strain. Plasmids were derived from the pGK and pATP vectors, in which the gene expression is controlled by the PGK1, ADH1, and TDH3 promoters [21 (link), 22 (link)].
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