C. neoformans strains H99 and Δafr3, Δafr1, and Δafr2 mutants were cultured in synthetic media (SM; 1.7 g yeast nitrogen base without amino acids (BD, Frankin Lakes, NJ, USA), 1 g drop out mix (USBiological Life Sciences, Salem, MA, USA), 4 mL ethanol, 5 g (NH4)2SO4, 3.3 g NaCl, 20 g glucose). Calorie restriction synthetic media was prepared as follows: CR, 1.7 g yeast nitrogen base without amino acids (BD, Frankin Lakes, NJ, USA), 1 g drop out mix (USBiological Life Sciences, Salem, MA, USA), 4 mL ethanol, 5 g (NH4)2SO4, 3.3 g NaCl, 0.5 g glucose. The Δafr3 strain was derived from the Madhani knockout collection, which is managed by the Fungal Genetics Stock Center. The Δafr1 and Δafr2 strains were gifted by Dr. Kwon-Chung Lab [14 (link)]. The mutant S. cerevisiae strains were cultured in complete supplement medium without uracil (CSM-Ura (MP Bio, Irvine, CA, USA); 20 g galactose, 1.7 g YNB, 5 g (NH4)2SO4, 0.77 g CSM-Ura). S. cerevisiae ADΔ strain, in which several ABC transporters and the URA3 locus were deleted, was previously described [20 (link)]. The ADΔ strain and pYES2 plasmid were obtained as a gift from Dr. Theodore C. White at the University of Missouri, Kansas City. All strains used in this study are maintained as 30% glycerol stocks and stored at −80 °C for future use.
Yeast nitrogen base without amino acids
Manufactured by BD
Sourced in United States, Japan, United Kingdom
Yeast nitrogen base without amino acids is a culture medium used for the growth and cultivation of microorganisms, particularly yeasts. It provides a defined source of nitrogen, vitamins, and minerals essential for microbial metabolism, excluding amino acids.
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Lab products found in correlation
Bacto peptone, by BD (7 mentions)
Yeast extract, by BD (6 mentions)
Peptone, by BD (5 mentions)
Uracil, by Merck Group (4 mentions)
Dextrose, by Thermo Fisher Scientific (3 mentions)
L arginine, by Merck Group (3 mentions)
L lysine, by Merck Group (3 mentions)
L tryptophan, by Merck Group (3 mentions)
L valine, by Merck Group (3 mentions)
L methionine, by Merck Group (3 mentions)
L histidine, by Merck Group (3 mentions)
L phenylalanine, by Merck Group (3 mentions)
L threonine, by Merck Group (3 mentions)
L isoleucine, by Merck Group (3 mentions)
L tyrosine, by Merck Group (3 mentions)
L leucine, by Merck Group (3 mentions)
Adenine hemisulfate, by Merck Group (3 mentions)
Yeast extract, by Nacalai Tesque (3 mentions)
Tryptone, by Nacalai Tesque (3 mentions)
Galactose, by Merck Group (3 mentions)
41 protocols using yeast nitrogen base without amino acids
1
Cryptococcus Mutant Strain Culture
2
Yeast Cell Wall N-Glycan Analysis
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S. cerevisiae strains were grown in YPD medium (1% yeast extract (Lab M, Heywood, UK), 2% peptone (BD, Franklin Lakes, NJ, USA), 2% glucose), minimal medium (0.67% yeast nitrogen base without amino acids (BD, Franklin Lakes, NJ, USA) supplemented with adenine and lysine) or SD medium (0.67 % yeast nitrogen base without amino acids supplemented with a dropout mix lacking leucine and uracil), using 2 % glucose or 1 % sorbitol. S. cerevisiae cultures were grown in shake flasks at 30 °C except for strains YMP05, YMP06, YMP07 and YMP09, which were grown at 28 °C. Cell densities of the liquid cultivations were monitored by measuring their optical density at 600 nm (OD600).
For cell wall protein N-glycan analysis, empty yeast strains were grown in YPD medium and collected in the midlog phase. Yeast strains containing plasmids were cultivated in SD medium containing 1 % raffinose, except for strains YMP15 and YMP16, which were cultivated in minimal medium containing 1 % raffinose. Expression of the glycosyltransferases GnTI and GnTII and the UDP-GlcNAc transporter KlMNN2-2 was induced at an OD600 of 1.0 using 2 % galactose, after which the cells were grown for 24h.
For cell wall protein N-glycan analysis, empty yeast strains were grown in YPD medium and collected in the midlog phase. Yeast strains containing plasmids were cultivated in SD medium containing 1 % raffinose, except for strains YMP15 and YMP16, which were cultivated in minimal medium containing 1 % raffinose. Expression of the glycosyltransferases GnTI and GnTII and the UDP-GlcNAc transporter KlMNN2-2 was induced at an OD600 of 1.0 using 2 % galactose, after which the cells were grown for 24h.
3
Yeast Strain Construction and Growth
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S. cerevisiae strains used in this study are listed in Table 1 . Yeast cells were grown aerobically at 26 °C in an orbital shaker (at 140 rpm), with a ratio of flask volume:medium volume of 5:1. The growth medium used was synthetic complete (SC) medium, containing drop-out, 2% (w/v) glucose (ThermoFisher Scientific, Waltham, MA, USA), and 0.67% (w/v) yeast nitrogen base without amino acids (BD BioSciences, San Jose, CA, USA), supplemented with appropriate amino acids or nucleotides [0.008% (w/v) histidine (Sigma Aldrich, St. Louis, MO, USA), 0.038% (w/v) methionine (Sigma Aldrich, St. Louis, MO, USA), 0.04% (w/v) leucine (Sigma Aldrich, St. Louis, MO, USA), and 0.008% (w/v) uracil (Sigma Aldrich, St. Louis, MO, USA)]. Deletion of AFT1 in ncr1Δ cells was performed using a deletion fragment containing HIS3 and the flanking regions of AFT1. Deletion of YCK3 in wild-type cells was performed using a deletion fragment containing KanMX4 and the flanking regions of YCK3. Yeast cells were transformed using the lithium acetate/single-stranded carrier DNA/PEG method as described [96 (link)]. Cells were selected by growing in a medium lacking histidine or containing geneticin (300 µg mL−1), and gene deletion was confirmed by standard PCR procedures.
4
Genotypes of Saccharomyces cerevisiae Strains
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The genotypes of Saccharomyces cerevisiae BY474167 (link), MC-F143 (link), and BY474267 (link) and the other recombinant strains used in this study are provided in Table 1 . The yeast strains were grown in YPD medium containing 1% (w/v) yeast extract, 2% peptone and 2% glucose or in SD medium containing 0.67% yeast nitrogen base without amino acids (BD Diagnostic Systems, Sparks, MD, USA) and 2% glucose. The SD medium was supplemented with amino acids and nucleotides (20 mg/L histidine, 60 mg/L leucine, 20 mg/L methionine, or 20 mg/L uracil), as required by the auxotrophic strains. Agar (2%; w/v) was added to the medium to produce YPD and SD solid media.
5
Yeast Strain Manipulation Protocol
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The parental wild-type yeast strain used in this study was BY4741. All gene deletions, genomic integrations, and plasmid transformations were done in this background using standard methods. For a complete list of strains used, see S1 Table .
Yeast cultures were grown at 30°C in YPD or synthetic defined (SD) media with appropriate nutrient dropouts. SD media used for growth of yeast cultures contained: 2% w/v dextrose (Thermo Fisher Scientific, Waltham, MA), 13.4 g/L Yeast Nitrogen Base without Amino Acids (BD Biosciences, San Jose, CA), 0.03 g/L L-isoleucine (Sigma-Aldrich, St. Louis, MO), 0.15 g/L L-valine (Sigma-Aldrich), 0.04 g/L adenine hemisulfate (Sigma-Aldrich), 0.02 g/L L-arginine (Sigma-Aldrich), 0.03 g/L L-lysine (Sigma-Aldrich), 0.05 g/L L-phenylalanine (Sigma-Aldrich), 0.2 g/L L-Threonine (Sigma-Aldrich), 0.03 g/L L-tyrosine (Sigma-Aldrich), 0.018 g/L L-histidine (Sigma-Aldrich), 0.09 g/L L-leucine (Sigma-Aldrich), 0.018 g/L L-methionine (Sigma-Aldrich), 0.036 g/L L-tryptophan (Sigma-Aldrich), and 0.018 g/L uracil (Sigma-Aldrich). Media additionally contained 5 mM or 10 mM guanidinium hydrochloride (Sigma-Aldrich) where indicated and bortezomib (LC Laboratories, Woburn, MA) treatment lasted 4 hours.
Yeast cultures were grown at 30°C in YPD or synthetic defined (SD) media with appropriate nutrient dropouts. SD media used for growth of yeast cultures contained: 2% w/v dextrose (Thermo Fisher Scientific, Waltham, MA), 13.4 g/L Yeast Nitrogen Base without Amino Acids (BD Biosciences, San Jose, CA), 0.03 g/L L-isoleucine (Sigma-Aldrich, St. Louis, MO), 0.15 g/L L-valine (Sigma-Aldrich), 0.04 g/L adenine hemisulfate (Sigma-Aldrich), 0.02 g/L L-arginine (Sigma-Aldrich), 0.03 g/L L-lysine (Sigma-Aldrich), 0.05 g/L L-phenylalanine (Sigma-Aldrich), 0.2 g/L L-Threonine (Sigma-Aldrich), 0.03 g/L L-tyrosine (Sigma-Aldrich), 0.018 g/L L-histidine (Sigma-Aldrich), 0.09 g/L L-leucine (Sigma-Aldrich), 0.018 g/L L-methionine (Sigma-Aldrich), 0.036 g/L L-tryptophan (Sigma-Aldrich), and 0.018 g/L uracil (Sigma-Aldrich). Media additionally contained 5 mM or 10 mM guanidinium hydrochloride (Sigma-Aldrich) where indicated and bortezomib (LC Laboratories, Woburn, MA) treatment lasted 4 hours.
6
Yeast Cultivation Protocols for Genetic Studies
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Details regarding Saccharomyces cerevisiae BY4741 [43] (link), MC-F1 [44] (link), BY4742 [43] (link) and other recombinant strains used in this study and their genotypes are provided in Tables 1 –3 . The yeast strains were grown in YPD medium containing 1% (w/v) yeast extract, 2% peptone and 2% glucose, or in SD medium containing 0.67% yeast nitrogen base without amino acids (BD-Diagnostic Systems, Sparks, MD, USA) and 2% glucose. Amino acids and nucleotides (20 mg/L histidine, 60 mg/L leucine, 20 mg/L methionine, or 20 mg/L uracil) were supplemented into SD medium as required by the auxotrophic strains. Agar (2%; w/v) was added to the medium to produce YPD and SD solid media.
7
Yeast and Bacterial Strain Cultivation Protocols
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The wild-type K. phaffii strain CBS7435 (NRRL-Y11430) was used in the REMI screen. The K. phaffii strain dnl4his433 (link) was used for accurate gene disruptions in the effective factor validation and multiple-disruption processes. E. coli strain DH5α was used for recombinant DNA manipulation.
K. phaffii strains were grown in YPD or YPG media [10 g/L yeast extract (Nacalai Tesque, Kyoto, Japan), 20 g/L Bacto peptone (BD Biosciences, San Jose, CA, USA) and 20 g/L glucose (for YPD) or 20 g/L glycerol (for YPG)], or in BMGY or BMMY media [10 g/L yeast extract, 20 g/L hipolypeptone (Nihon Pharmaceutical, Tokyo, Japan), 13.4 g/L yeast nitrogen base without amino acids (BD Biosciences), 0.4 mg/L biotin (Nacalai Tesque), 100 mM potassium phosphate buffer (final, pH 6.0) and 20 g/L glycerol (for BMGY) or 20 g/L methanol (for BMMY)]. E. coli strains were grown in LB media with 5 g/L yeast extract, 10 g/L tryptone (Nacalai Tesque) and 5 g/L NaCl, supplemented with ampicillin (100 μg/mL). YPD agar plate contained 20 g/L agar in YPD media with antibiotics (500 μg/mL G418 (Wako Pure Chemical Industries, Osaka, Japan), 100 μg/mL Zeocin (InvivoGen, San Diego, CA, USA), 100 μg/mL hygromycin (Wako Pure Chemical Industries), 50 μg/mL nourseothricin (Werner BioAgents, Jena, Germany), and/or 100 μg/mL blasticidin (Wako Pure Chemical Industries)). Square YPD plates contained 100 μg/mL Zeocin.
K. phaffii strains were grown in YPD or YPG media [10 g/L yeast extract (Nacalai Tesque, Kyoto, Japan), 20 g/L Bacto peptone (BD Biosciences, San Jose, CA, USA) and 20 g/L glucose (for YPD) or 20 g/L glycerol (for YPG)], or in BMGY or BMMY media [10 g/L yeast extract, 20 g/L hipolypeptone (Nihon Pharmaceutical, Tokyo, Japan), 13.4 g/L yeast nitrogen base without amino acids (BD Biosciences), 0.4 mg/L biotin (Nacalai Tesque), 100 mM potassium phosphate buffer (final, pH 6.0) and 20 g/L glycerol (for BMGY) or 20 g/L methanol (for BMMY)]. E. coli strains were grown in LB media with 5 g/L yeast extract, 10 g/L tryptone (Nacalai Tesque) and 5 g/L NaCl, supplemented with ampicillin (100 μg/mL). YPD agar plate contained 20 g/L agar in YPD media with antibiotics (500 μg/mL G418 (Wako Pure Chemical Industries, Osaka, Japan), 100 μg/mL Zeocin (InvivoGen, San Diego, CA, USA), 100 μg/mL hygromycin (Wako Pure Chemical Industries), 50 μg/mL nourseothricin (Werner BioAgents, Jena, Germany), and/or 100 μg/mL blasticidin (Wako Pure Chemical Industries)). Square YPD plates contained 100 μg/mL Zeocin.
8
Screening Transformants on OBR-HEC Agar
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Transformants were screened on Ostazin brilliant red hydroxyethyl cellulose (OBR-HEC) agar plates (6.7 g/L yeast nitrogen base without amino acids [BD-Diagnostic Systems, Sparks, MD], 100 g/L glucose, 10 g/L OBR-HEC and 20 g/L agar). Spores were transferred to the OBR-HEC, agar plates, and the development of the clearing zone was monitored for 24 hr.
9
Yeast Growth Conditions and Strains
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Details of the yeast S. cerevisiae strains BY474151 (link), BY474251 (link), MC-F144 (link) and other recombinant strains used in this study and their genotypes are outlined in Table 3 . The yeast strains were grown in YPD medium containing 1% (w/v) yeast extract, 2% peptone and 2% glucose or in SD media containing 0.67% yeast nitrogen base without amino acids (BD-Diagnostic Systems, Sparks, MD, USA) and containing 2% glucose. Amino acids and nucleotides (20 mg/L histidine, 60 mg/L leucine, 20 mg/L methionine, or 20 mg/L uracil) were supplemented into SD media lacking the relevant auxotrophic components. Agar (2%; w⁄v) was added to the medium described above to produce YPD and SD solid media.
10
Yeast Protein Immunoblotting Reagents
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The following antibodies were from Sigma-Aldrich: glucose-6-phosphate dehydrogenase (G6PDH), HA-peroxidase, and FLAG M2. Antibodies for ubiquitin (P4D1) and VCP/Cdc48 were from Cell Signaling Technology. The antibody for Por1 was from Thermo Fisher Scientific. The GFP antibody was obtained from CWbio.
Yeast extract, peptone, and yeast nitrogen base without amino acids were purchased from BD. Yeast complete supplement mixture was purchased from MP Biomedicals. Yeast amino acid dropout supplements were obtained from Takara Bio Inc. CHX was obtained from Amresco. MG132 was from EMD Millipore. Other chemicals or reagents were obtained from Sigma-Aldrich if not otherwise indicated.
Yeast extract, peptone, and yeast nitrogen base without amino acids were purchased from BD. Yeast complete supplement mixture was purchased from MP Biomedicals. Yeast amino acid dropout supplements were obtained from Takara Bio Inc. CHX was obtained from Amresco. MG132 was from EMD Millipore. Other chemicals or reagents were obtained from Sigma-Aldrich if not otherwise indicated.
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