As described in Searle et al.,13 (link)S. cerevisiae strain BY4741 (Dharmacon) was cultured at 30 °C in YEPD and harvested at the mid-log phase. Cells were pelleted and lysed in a buffer of 8 M urea, 50 mM Tris (pH 8), 75 mM NaCl, 1 mM EDTA (pH 8) followed by seven cycles of 4 min bead beating with glass beads. After a 1 min rest on ice, lysate was collected by piercing the tube and centrifuging for 1 min at 3000 × g and 4 °C into an empty eppendorf. After further centrifugation at 21,000 × g and 4 °C for 15 min, the protein content of the supernatant was removed and estimated using BCA. Proteins were then reduced with 5 mM dithiothreitol for 30 min at 55 °C, alkylated with 10 mM iodoacetamide in the dark for 30 min at room temperature, and diluted to 1.8 M urea, before digestion with sequencing-grade trypsin (Pierce) at a 1:50 enzyme-to-substrate ratio for 16 h at 37 °C. In all, 5 N HCl was added to approximately pH 2 to quench the digestion, and the resulting peptides were desalted with 30 mg MCX cartridges (Waters). Peptides were dried with vacuum centrifugation and brought to 1 μg/3 μl in 0.1% formic acid (buffer A) prior to MS acquisition. All measurements of yeast were performed on the same biological replicate to assess technical variability in the method.
By4741
Manufactured by Horizon Discovery
Sourced in United Kingdom
BY4741 is a yeast strain used as a model organism in genetic and molecular biology research. It is a haploid Saccharomyces cerevisiae strain that is commonly used as a reference strain. The strain's genotype is MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0.
Automatically generated - may contain errors
Lab products found in correlation
Mcx cartridges, by Waters Corporation (1 mentions)
Pyes2, by Thermo Fisher Scientific (1 mentions)
Yeastmaker yeast transformation system 2 kit, by Takara Bio (1 mentions)
By4742, by Horizon Discovery (1 mentions)
Nico21 de3, by New England Biolabs (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
10 protocols using by4741
1
Yeast Proteome Extraction and Trypsin Digestion
2
Total RNA Extraction from Yeast and Plasmid Isolation
3
Verification of Yeast Knockout Strains
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All S. cerevisiae yeast knockout (YKO) strains used in this study were derived from the haploid parental WT strain BY4741 (Mata his3∆1 leu2∆0 met 15∆0 ura3∆0) and were purchased from Dharmacon (Lafayette, CO). The identity of each YKO strain was verified by PCR, using strain‐specific primers. The primer sequences and PCR product sizes for the YKO strains were obtained from the Saccharomyces Genome Deletion Project website (http://www-sequence.stanford.edu/group/yeast_deletion_project/downloads.html # instru). The WT and mutant PDR5 yeast transformants used in this study are isogenic and were derived from the ∆pdr5 YKO strain. The identity of each transformant was verified by sequence analysis. Yeast strains were streaked on appropriate growth medium as follows: BY4741, on yeast peptone dextrose (YPD) agar; the YKO strains, on YPD agar with 200 μg/mL G418; and the ∆pdr5 transformants, on synthetic dropout agar lacking uracil (SD‐Ura), and incubated at 30°C until individual colonies formed. Several colonies from each PDR5 mutant transformant were restreaked onto appropriate fresh agar media and incubated at 30°C to obtain pure clonal isolates.
4
Complementation of Yeast bts1Δ Mutant
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The yeast gene deletion mutant strain BY4741 bts1Δ (YPL069C) and the WT strain BY4741 (MATa his3Δ1, leu2Δ0 met15Δ0 ura3Δ0) were purchased from Dharmacon (Cambridge, United Kingdom) and preserved in 30% glycerol. The full-length PsBTS1 sequence was cloned into pYES2 (Invitrogen, Carlsbad, CA, United States) and transformed into the BY4741 bts1Δ mutant strain using a Yeastmaker Yeast Transformation System 2 kit (Takara, Dalian, China) following the manufacturer’s protocol. The empty plasmid pYES2 was transformed into yeast WT strain BY4741 as well as the bts1Δ mutant, which served as the controls. Colonies grew on Synthetic Defined medium lacking uracil containing glucose were further screened by PCR. The transformed yeast cells were cultured overnight in YPD broth, then rinsed with water to remove YPD completely. The yeast cell solution was adjusted to an optical density at 600 nm of 0.2, then serially diluted 10 times. A 5-μl drop from each dilution was spotted into YPD medium without uracil, containing either glucose or galactose, and grown at 14, 25, or 30°C for 7 days.
5
Yeast-based Protein Expression and Validation
6
Homologous Recombination for Yeast Strain Construction
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All strains from this study were made by homologous recombination as described (Longtine et al., 1998 (link); Janke et al., 2004 (link); Sheff and Thorn, 2004 (link)). BY4741 and BY4742 yeast strains were purchased from GE Dharmacon (Lafayette, CO). Strains were propagated in rich medium (YPD: 1% yeast extract, 2% peptone, 2% dextrose) or SD minimal medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% synthetic complete mix, 2% dextrose) supplemented with the appropriate amino acids for plasmid selection. Plasmids were made by homologous recombination in yeast, rescued in Escherichia coli, and confirmed by sequencing. pHPH was made by PCR amplification of the hygromycin resistance gene HPH from pFA6a-hphNT1 and cotransformation of the PCR product with linearized pRS416. Proteins were tagged with either the bright GFP variant GFP+ (Scholz et al., 2000 (link)) or GFP(envy) (Slubowski et al., 2015 (link)), a gift from C. Slubowski (University of Massachusetts, Boston, MA), by PCR amplification of the GFP::HIS cassette and transformation into yeast. Strains and plasmids are described in Supplemental Tables S1 and S2.
7
Yeast Stress Response Protocol
8
Yeast Strain Cultivation and Storage
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Glycerol stocks of [PRION+], [prion−], BY4741, Swi1 DAmP and deletion yeast strains (Horizon Discovery, Waterbeach UK) were stored at −80 °C. Prior to all experiments, [PRION+], [prion−] and BY4741 yeast were streaked onto YPD and incubated at 30 °C for 2–3 days. Similarly, Swi1 DAmP and deletion strains were streaked onto YPD + 200 μg/mL G418 and incubated at 30 °C for 2–3 days. Strains were then inoculated into YPD liquid media (or YPD + 200 μg/mL G418 for Swi1 DAmP and deletion strains) and grown to saturation overnight at 30 °C with shaking at 200 rpm. Liquid cultures were then standardized to an OD600 of 0.3 and allowed to grow for 5 h at 30 °C at 200 rpm until an OD600 of 0.6–0.8 was reached [67 (link)]. Cells were then pelleted, flash frozen, and stored at −80 °C.
9
Preparation of Selective Growth Media for Genetic Studies
10
Cloning and Expression of S. cerevisiae Arl1 and Gea2
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The gene sequences encoding S. cerevisiae Arl1 and Gea2 were PCR-amplified from genomic DNA of S. cerevisiae BY4741 obtained from Horizon Discovery (Waterbeach, UK) with pairs of primers 5′-GTCTCTCCCATGGGTAACATTTTTAGTTCAATGTTTG-3′/5′-GGTTCTCCCCAGCTAACTGTTCCTCTTTTATAA-3′ and 5′-TACTTCCAATCCAATGCAATGAGTGATAGGGAATTCGTC-3′/5′-TTATCCACTTCCAATGCCTTAATCCTTTTCTACATCAGATAACTTC-3′, respectively. The genes were inserted using ligation-independent cloning into vectors acquired from the DNASU Plasmid Repository (Tempe, AZ), pMCSG28 for the expression of Arl1 and pMCSG21 for the expression of Gea2. Using Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA), expression vectors for Q72L Arl1 and also an N-terminal 17-residue truncated Q72L Arl1 (termed “ΔNT17 Q72L Arl1”) were also generated. After DNA sequence verification (GENEWIZ, South Plainfield, NJ), the E. coli strain NiCo21(DE3) (New England Biolabs, Ipswich, MA) was transformed with constructed vectors. To overcome codon bias, an additional plasmid pRARE2 isolated from E. coli strain Rosetta 2 (Novagen, Madison, Wisconsin) was also included in the transformation for Gea2 expression. The expressed protein Arl1 had a C-terminal 6xHis-tag, and Gea2 had an N-terminal 6xHis-tag to facilitate their purifications individually.
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