The strains used in this study are shown in Table 1 . Starmerella bombicola KSM36, an industrial strain for sophorolipid production (Inoue and Ito 1982 ), ura3-auxotrophic mutant KSM-ura3Δ and FAO1-negative mutant KSM-fao1Δ (Takahashi, Igarashi and Hagihara 2016 (link)) were used as the parent strains. The methods for plasmid maintenance, cultivation of S. bombicola and preparation of cell extracts were reported earlier without hygromycin (Takahashi, Igarashi and Hagihara 2016 (link)). To obtain hygromycin resistant strains, 0.05% hygromycin B (FUJIFILM Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to the synthetic dextrose (SD) medium (0.67% yeast nitrogen base without amino acids (Sigma-Aldrich, St. Louis, MO, USA) and 2% glucose).
Yeast nitrogen base without amino acids
Manufactured by Merck Group
Sourced in United States
Yeast nitrogen base without amino acids is a microbial growth medium that provides a source of nitrogen, vitamins, and other nutrients essential for the cultivation of a variety of microorganisms, including yeast. It is a defined and standardized medium that does not contain amino acids, allowing for the study of amino acid synthesis and other metabolic processes in the target organisms.
Automatically generated - may contain errors
Lab products found in correlation
L lysine 13c615n2, by Cambridge Isotopes (2 mentions)
Yeast synthetic dropout without lysine, by Merck Group (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Dextrose, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Hygromycin b, by Fujifilm (1 mentions)
Potassium phosphate monobasic, by Merck Group (1 mentions)
Succinic acid, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Ammonium sulfate, by Merck Group (1 mentions)
Oleate, by Merck Group (1 mentions)
P9417, by Merck Group (1 mentions)
O1008, by Merck Group (1 mentions)
Glass bead, by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Honeywell (1 mentions)
Itraconazole, by Selleck Chemicals (1 mentions)
Lovastatin, by Selleck Chemicals (1 mentions)
Uniconazole, by Selleck Chemicals (1 mentions)
20 protocols using yeast nitrogen base without amino acids
1
Construction of Hygromycin-resistant Starmerella
2
Yeast-based Screening of GLA Variants
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Yeast (INVSc1) cells (Catalog #C81000, ThermoFisher Scientific), transformed with vectors expressing GLA variants using the lithium acetate procedure65 (link), were plated on SD-Ura medium (2 g/L SD Media—Ura, 6.8 g/L yeast nitrogen base without amino acids [Sigma Aldrich]), 3.06 g/L sodium dihydrogen phosphate, 0.804 g/L disodium hydrogen phosphate (pH 6.0) 6% glucose. After 72 h incubation at 30 °C, colonies were picked into the wells of Axygen® 1.1 mL 96-well deep well plates containing 200 µL/well SD-Ura. The cells were grown for 20–24 h in a Kuhner shaker (250 rpm, 30 °C, and 85% relative humidity). Overnight culture samples (20 µL) were transferred into Coming Costar® 96-well deep plates filled with 380 µL of SD-Ura broth supplemented with 2% glucose. The plates were incubated for 66–84 h in a Kuhner shaker (250 rpm, 30 °C, and 85% relative humidity). The cells were then pelleted (4000 rpm × 20 min), and the supernatants isolated for immediate analysis, or stored at 4 °C prior to analysis.
3
Recombinant MVP Protein Production
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For the production of the MVP protein, the vacuolar aspartyl protease PEP4-deficient K. phaffii SMD1168 (his4, ura3, pep4::URA3) strain was used [39 ]. The strain is available from Invitrogen (Carlsbad, CA, USA). K. phaffii was grown in a flask in BMDY medium (10 g/L yeast extract, 20 g/L bacto peptone, 20 g/L dextrose, 100 mM potassium phosphate buffer, pH 5.8, 13.4 g/L yeast nitrogen base without amino acids, 0.4 mg/L biotin) [40 ,41 (link)]. All media were from Biolife; monobasic potassium phosphate, yeast nitrogen base without amino acids and biotin were from Sigma-Aldrich. Standard liquid and plate growth were performed on YPD medium (10 g/L yeast extract, 20 g/L bacto peptone, 20 g/L dextrose, 20 g/L agar omitted in liquid medium) with or without 100 μg/mL zeocin. After electroporation, YPDS plates containing 1 M sorbitol and 100 µg/mL Zeocin were used for the selection of the transformants.
4
Yeast Culture Media Preparation and Selection
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YPD media: 20 g/L Bacto™ Peptone, 10 g/L Bacto™ Yeast extract, 20 g/L glucose.
Synthetic complete (SC) meda without uracil: 1.92 g/L Yeast Synthetic Drop-out Media Supplements without uracil (Sigma-Aldrich Co. LLC. Catalog number Y1501), 6.7 g/L Yeast Nitrogen Base Without Amino Acids (Sigma-Aldrich Co. LLC. Catalog number Y0626), 20 g/L glucose. Feed-In-Time (FIT) was based on EnPump200 (Enpresso GmbH), and made according to protocol enclosed with the product. Agar plates: SC media including agar (15 g/L).
Uracil auxotrophy in parent strains was introduced by selecting for lack of URA3 function on agar plates of SC medium without uracil containing also 5-fluoroorotic acid (5-FOA, 0.74 g/L) and uracil (30 mg/L). Yeast transformants were isolated on SC without uracil agar plates.
Synthetic complete (SC) meda without uracil: 1.92 g/L Yeast Synthetic Drop-out Media Supplements without uracil (Sigma-Aldrich Co. LLC. Catalog number Y1501), 6.7 g/L Yeast Nitrogen Base Without Amino Acids (Sigma-Aldrich Co. LLC. Catalog number Y0626), 20 g/L glucose. Feed-In-Time (FIT) was based on EnPump200 (Enpresso GmbH), and made according to protocol enclosed with the product. Agar plates: SC media including agar (15 g/L).
Uracil auxotrophy in parent strains was introduced by selecting for lack of URA3 function on agar plates of SC medium without uracil containing also 5-fluoroorotic acid (5-FOA, 0.74 g/L) and uracil (30 mg/L). Yeast transformants were isolated on SC without uracil agar plates.
5
Yeast Tetrad Dissection and SILAC Experiments
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Tetrad dissections were performed on standard YPD plates and incubated at 30°C for 72 h. Yeast strains were grown according to normal growth conditions. For spotting assays, myriocin or Aureobasidin A were added at concentrations as indicated and the plates were grown at 30°C for 48 h. For SILAC experiments SDC medium without lysine with 2% glucose, 6.7 g/liter yeast nitrogen base without amino acids and yeast synthetic dropout without lysine (Sigma-Aldrich) was used. Normal or heavy lysine (L-lysine 13C615N2; Cambridge Isotope Laboratories) at the concentration of 30 g/liter were added. Cells were diluted from an overnight pre-culture to an OD600 = 0.2 and grown at 30°C. Cells were harvested at an OD600 = 1.0–1.4.
6
Yeast Genetic Manipulation and Protein Expression
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All yeast strains used in this study are detailed in Table S1. Yeast genetic manipulations were conducted using classical yeast knock-in/out protocols. For ectopic protein expression, genes were cloned into pBP73-C or -G vectors encoding either the GPD promoter or CPY promoter. All plasmids used in this study are detailed in Table S2. Yeast transformations were performed using the lithium acetate method. We used Ds-Red HDEL to label the ER. Strains were selected using antibiotics or dropout selection media. All chemicals used to make the yeast media were purchased from Sigma-Aldrich (succinic acid, sodium hydroxide, ammonium sulfate, yeast nitrogen base without amino acids, or ammonium sulfate). Yeast media was supplemented with a final concentration of 2% dextrose unless otherwise indicated. oleate (Sigma-Aldrich; O1008) and POA (Sigma-Aldrich; P9417) were added to the culture media as indicated (0.2% oleate is equivalent to 6.32 mM, and 0.2% POA is equivalent to 7.04 mM).
7
Comprehensive Analytical Reagents Protocol
8
Yeast Signaling Pathway Assay
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Polyoxyethylene-10-laurylether (C12E10), L-α-phosphatidylcholine (P5638), ATP (disodium salt, vanadium-free), SDS, yeast synthetic drop-out media supplement without leucine, yeast nitrogen base without amino acids, dextrose, enzymes, and cofactors were obtained from Sigma. Tryptone and yeast extract were from Difco and the [γ-32P]-ATP was from PerkinElmer Life Sciences (Boston, MA). Salts and reagents were of analytical reagent grade.
9
Yeast Growth and Genetic Manipulation
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Yeast strains were grown under standard conditions. For rapamycin spotting assays, the cells were grown in YPD (2% glucose, 2% peptone, and 1% yeast extract) and the drug added in a final concentration of 4 ng/ml. Plates were incubated for 48 h at 30°C.
For SILAC labeling, yeast cells were grown in SDC–lysine medium (2% glucose, 6.7 g/liter yeast nitrogen base without amino acids and 1.92 g/liter yeast synthetic dropout without lysine; Sigma Aldrich). Precultures were grown over day at 30°C in the presence of 30 mg/liter normal lysine or heavy lysine (L-lysine 13C615N2; Cambridge Isotope Laboratories), diluted in the evening and cells grown to the log phase in the next morning before harvesting.
For microscopy experiments, cells were grown in SDC–lysine medium with the addition of 30 mg/liter normal lysine. The Mup1 uptake assay was performed in SDC–methionine medium, inducing Mup1 endocytosis by the addition of 1 mM methionine.
For tetrad dissections, diploid cells generated from mating of haploid deletion mutants in YPD were sporulated on sporulation plates (3% agar, 1% KOAc) for 5 d at 30°C.
For SILAC labeling, yeast cells were grown in SDC–lysine medium (2% glucose, 6.7 g/liter yeast nitrogen base without amino acids and 1.92 g/liter yeast synthetic dropout without lysine; Sigma Aldrich). Precultures were grown over day at 30°C in the presence of 30 mg/liter normal lysine or heavy lysine (L-lysine 13C615N2; Cambridge Isotope Laboratories), diluted in the evening and cells grown to the log phase in the next morning before harvesting.
For microscopy experiments, cells were grown in SDC–lysine medium with the addition of 30 mg/liter normal lysine. The Mup1 uptake assay was performed in SDC–methionine medium, inducing Mup1 endocytosis by the addition of 1 mM methionine.
For tetrad dissections, diploid cells generated from mating of haploid deletion mutants in YPD were sporulated on sporulation plates (3% agar, 1% KOAc) for 5 d at 30°C.
10
S. cerevisiae and E. coli Growth Conditions
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The S.
cerevisiae strain used was BY4741 (MATa his3Δ1
leu2Δ0 met15Δ0 ura3Δ0). For selection, cells were
grown at 30 °C in minimal synthetic media or agar plates containing
6.8 g/L yeast nitrogen base without amino acids (Sigma-Aldrich), 2
g/L dextrose (Formedium), and a complete supplement mixture of amino
acids minus leucine or minus leucine and histidine (MP Biomedicals). E. coli DH5α cells were used for all cloning
steps and were grown at 37 °C on Lysogeny Broth (LB) agar plates
or in liquid LB media with shaking at 200 rpm. 35 μg/mL chloramphenicol
or 50 μg/mL carbenicillin was added as appropriate.
cerevisiae strain used was BY4741 (MATa his3Δ1
leu2Δ0 met15Δ0 ura3Δ0). For selection, cells were
grown at 30 °C in minimal synthetic media or agar plates containing
6.8 g/L yeast nitrogen base without amino acids (Sigma-Aldrich), 2
g/L dextrose (Formedium), and a complete supplement mixture of amino
acids minus leucine or minus leucine and histidine (MP Biomedicals). E. coli DH5α cells were used for all cloning
steps and were grown at 37 °C on Lysogeny Broth (LB) agar plates
or in liquid LB media with shaking at 200 rpm. 35 μg/mL chloramphenicol
or 50 μg/mL carbenicillin was added as appropriate.
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