All of the C. albicans strains used in this study were derived from strain SC5314, and a complete list of the strains is provided in Table S2 . nourseothricin-sensitive C. albicans strains were cultured in yeast extract-peptone-dextrose (YPD) liquid medium at 30°C and harvested at an optical density at 600 nm between 0.5 and 0.8 prior to transformation by a modified version of the standard lithium acetate protocol (10 ); see our detailed protocol in Text S1 . After recovery in liquid YPD for 5 h, nourseothricin-resistant transformants were selected on YPD agar supplemented with 200 µg/ml nourseothricin (GoldBio). Subsequent removal of the CRISPR components was performed by single-colony isolation on synthetic defined (SD) agar medium minus leucine for the LEUpOUT method or by culturing overnight in YP-maltose liquid medium, followed by screening on YPD agar supplemented with 25 µg/ml nourseothricin for the FLP recombinase-mediated method (see Text S1 for details). The generation of homozygous URA3 deletion strains was confirmed by patching to SD minus uracil versus YPD plates; both medium types were supplemented with 200 µg/ml nourseothricin to maintain selection for strains that had integrated the CRISPR components. All E. coli strains were derived from DH5α and cultured at 37°C in LB medium supplemented with 100 µg/ml carbenicillin.
Nourseothricin
Manufactured by GoldBio
Sourced in United States
Nourseothricin is a selection marker for use in bacterial and eukaryotic cells. It confers resistance to the antibiotic nourseothricin, allowing for the selection of cells that have successfully integrated the desired genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Phleomycin, by InvivoGen (2 mentions)
Gene pulser cuvette, by Bio-Rad (2 mentions)
Hygromycin b, by Cayman Chemical (2 mentions)
Geneticin g418, by GoldBio (2 mentions)
Zeocin, by Cayman Chemical (2 mentions)
Eporator, by Eppendorf (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Q5 reaction buffer, by New England Biolabs (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Nourseothricin, by Promega (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Bmgy buffered glycerol complex medium, by Teknova (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Difco sabouraud dextrose agar, by BD (1 mentions)
Ec plan neofluar 63 1.25 oil objective, by Zeiss (1 mentions)
M1000, by Tecan (1 mentions)
G418 sulfate, by Sangon (1 mentions)
Hygromycin b, by Roche (1 mentions)
20 protocols using nourseothricin
1
Construction of C. albicans Knockout Strains
2
Genetic Manipulation of Fission Yeast
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Strains were grown in yeast extract with supplements (YES) or Edinburgh Minimal Media (EMM), as indicated. Tag integration and knockouts were generated with a previously described PCR-based strategy and verified by PCR and western blotting55 (link) (primer sequences in Supplementary Table 3 ). Protein A-tagged strains were generated according to ref. 56 (link). The bmc1∆ strain was constructed by replacing the bmc1 open reading frame with the phleomycin resistance cassette and flanking primers containing 750 nucleotides of homology to the bmc1 genomic locus. Correct genotypes were selected on YES plates with the corresponding antibiotic (200 μg/mL G418, Sigma; 100 μg/mL Nourseothricin, GoldBio; 100 μg/mL phleomycin, Invivogen). Other strains were created by mating and antibiotic selection. A list of strains is provided in Supplementary Table 4 .
3
Yeast Strain Cultivation Protocol
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BSGX001 was cultured at 30 °C in synthetic complete dropout uracil (SC-Ura) medium [1.7 g L−1 yeast nitrogen base, 5 g L−1 ammonium sulfate, and 0.77 g L−1 CSM-Ura (Sunrise Science Products, USA)] supplemented with 20 g L−1 glucose as the carbon source. The derived strains were also cultured in SC-Ura supplemented with 20 g L−1 glucose, but G418 (Genview, China) (200 mg L−1 in liquid medium and 600 mg L−1 in solid medium) and nourseothricin (Gold Biotechnology, USA) (100 mg L−1 in liquid medium and 200 mg L−1 in solid medium) were supplemented as necessary. The fermentation medium YNB (pH 5.5) was composed of 1.7 g L−1 yeast nitrogen base and 5 g L−1 ammonium sulfate, 20 g L−1 glucose, and 20 g L−1 xylose were used as the carbon source. To turn off the CTR1 promoter, 300 μM CuSO4 was included in the fermentation broth at the designated time.
4
Isolation and Characterization of Antibiotic-Resistant Strains
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Single ribosomal operon strain, SQ110, was grown overnight in CAMHB at 37°C and resuspended in one-fifth volume in fresh medium. Thereafter, 200 μL of the suspension was plated on selective LB agar plates containing nourseothricin (GoldBio, St Louis, MO) at 32-, 64-, and 128-fold the MIC of the parent SQ100 strain and incubated for 48 to 72 hours at 35°C in ambient air until mutant colonies became visible. Selected colonies were passaged on plates with matching concentrations of nourseothricin for further analysis. Aminoglycoside resistance mutants were isolated in a similar manner selecting on apramycin.
Genomic DNA from nourseothricin-resistant strains was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) from 3 mL of overnight culture in CAMHB containing nourseothricin (32 μg/mL, 64 μg/mL, and 128 μg/mL). Subsequent genomic DNA was amplified using primers F16S+23S, R16S+23S, and 16SR (S11 Table ), with primer 16SR as previously described [72 (link)]. All amplification reactions were performed using Q5 high-fidelity DNA polymerase, Q5 reaction buffer, and DNTPs from New England Biolabs (Ipswich, MA) using a melting temperature of 68°C, an annealing temperature of 65°C, and an extension time of 2 minutes and 45 seconds. PCR products were purified using the QIAquick Purification Kit. Sanger sequencing was performed to identify mutations.
Genomic DNA from nourseothricin-resistant strains was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) from 3 mL of overnight culture in CAMHB containing nourseothricin (32 μg/mL, 64 μg/mL, and 128 μg/mL). Subsequent genomic DNA was amplified using primers F16S+23S, R16S+23S, and 16SR (
5
Recombinant Pichia pastoris Expressing Rotavirus Antigens
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Wild-type Komagataella phaffii (NRRL Y-11430) was modified to express variants of P[4], P[6], or P[8] under control of the AOX1 promoter using a commercial vector (pPICZ A, Thermo Fisher Scientific). Modified vectors containing markers for selection on G418 (Thermo Fisher Scientific) or Nourseothricin (Gold Biotechnology) were also used to generate a single strain expressing all three serotypes.
Strains for initial characterization were grown in 3 mL culture in 24-well deep well plates (25 °C, 600 rpm) using complex medium (BMGY-Buffered Glycerol Complex Medium, Teknova) supplemented to 4% (v/v) glycerol. After 24 h of biomass accumulation, cells were pelleted and resuspended in BMMY (Buffered Methanol Complex Medium, Teknova) containing 1.5% (v/v) methanol. Samples for RNA-seq and ribosome profiling were collected after 16 h growth in BMMY.
Strains were additionally cultivated in 200 mL culture in 1 L shake flasks to generate material for non-clinical studies and in InSCyT [36 ] bioreactors for demonstration of end-to-end manufacturing. At both scales, cells were grown in rich defined medium [53 (link)] supplemented to 4% (v/v) glycerol for biomass accumulation or 5% (v/v) methanol for production. In the bioreactor, temperature, pH, and dissolved oxygen were maintained at 25 °C, 6.5, and 25%, respectively.
Strains for initial characterization were grown in 3 mL culture in 24-well deep well plates (25 °C, 600 rpm) using complex medium (BMGY-Buffered Glycerol Complex Medium, Teknova) supplemented to 4% (v/v) glycerol. After 24 h of biomass accumulation, cells were pelleted and resuspended in BMMY (Buffered Methanol Complex Medium, Teknova) containing 1.5% (v/v) methanol. Samples for RNA-seq and ribosome profiling were collected after 16 h growth in BMMY.
Strains were additionally cultivated in 200 mL culture in 1 L shake flasks to generate material for non-clinical studies and in InSCyT [36 ] bioreactors for demonstration of end-to-end manufacturing. At both scales, cells were grown in rich defined medium [53 (link)] supplemented to 4% (v/v) glycerol for biomass accumulation or 5% (v/v) methanol for production. In the bioreactor, temperature, pH, and dissolved oxygen were maintained at 25 °C, 6.5, and 25%, respectively.
6
Tagging Septin Ring with GFP
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To tag with green fluorescent protein (GFP) the septin ring as has been previously described (35 (link)), GFP was inserted in frame at the C terminus of CDC10 by using a nourseothricin selection method (36 (link)). Briefly, the GFP-NAT1 cassette was amplified from plasmid pGFP-NAT1 using primers CDC10-GFP-5DR and CDC10-GFP-3DR; transformation of the cassette into THE1-CIp10 and tSEC6 strains was completed using the lithium acetate method, with a 4-h growth step in YPD added after the heat shock step to allow integration and translation of the NAT1 gene before exposing the cells to nourseothricin, as described previously (36 (link)). Transformants were selected on Difco Sabouraud-dextrose agar (BD, Franklin Lakes, NJ) containing 200 μg/ml nourseothricin (Gold Biotechnology, St. Louis, MO). Primers flanking the CDC10 open reading frame (Table 2 ) were used to screen for transformants carrying the CDC10-GFP allele. DIC and GFP fluorescence images were acquired after induction of filamentation in buffered RPMI for 6 and 24 h in the presence or absence of DOX and in yeast cells after 24 h in YPD with or without DOX. DIC and GFP fluorescence images were acquired using AxioVision 4.7 software (Carl Zeiss AG, Jena, Germany) and a Zeiss EC Plan-NeoFluar 63×/1.25× oil objective (Carl Zeiss AG, Jena, Germany).
7
Cytotoxicity Assay of Compounds
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J774A.1 mouse macrophage and LLC-PK1 porcine renal tubule epithelial cell lines were plated in 384-well tissue culture dishes with white walls and clear bottoms (Greiner, Monroe, NC, catalog #781098) at approximately 7.0 × 105 cells/cm2 and approximately 7.0 × 104 cells/cm2, respectively, in M199 medium lacking phenol red supplemented with 3% porcine serum and a final concentration of 125 nM SYTOX Green. Approximately 1 day after plating (at 50% to 75% confluence), the cell lines were treated with 2-fold doubling dilutions of nourseothricin (Gold Biotechnology), S-D, and S-F, dispensed with the HP D300 digital dispensing system (HP, Palo Alto, CA) using Tween-20 as the surfactant. Microplates were vortexed for 30 seconds to ensure thorough mixing within the wells prior to incubation. SYTOX Green fluorescence was read with a TECAN M1000 multi-mode reader using excitation 485 ± 7 nm and emission 535 ± 10 nm settings at indicated time points with otherwise continuous incubation at 37°C with 5% CO2 for 5 days.
8
Yeast Strain Construction and Genetic Manipulation
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Yeast strains used and constructed in this study are listed in Supplemental Table S1 and were grown at 30°C on YEPD (2% dextrose, 2% peptone, 1% yeast extract) except where noted. Transformations of PCR products and plasmids into yeast cells were carried out using the lithium acetate method according to Ito et al. (1983) (link). Yeast strains in which the ULM of Hsh155 is mutated (Hsh155RW-DA) were constructed by CRISPR/Cas9-mediated editing of the endogenous HSH155 using a modified approach from that described (DiCarlo et al. 2013 (link)) (see below). The CUS2 ORF was deleted from start to stop codon by transformation and integration of a natNT2 PCR product with ends homologous to sequences flanking the CUS2 ORF and conferring resistance to 100 µg/mL nourseothricin (GoldBio) as described (Janke et al. 2004 (link)). Strains expressing C-terminal Lea1-TAP were generated by PCR of sequences encoding the TAP-tag and the kanMX4 selectable marker, transformation, and selection on plates supplemented with 200 µg/mL G418 (Sigma-Aldrich) as described in Janke et al. (2004) (link) and Rigaut et al. (1999) (link).
9
CRISPR-Mediated Gene Editing in Yeast
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20 μL of prepared cells, 1–3 μg of drug resistant cassette DNA, CRISPR mix, and RNAse free water to a final volume of 45 μL was mixed and transferred to a BioRad Gene Pulser cuvette (0.2 cm gap). Cells were pulsed using an Eppendorf Eporator at 1500 V and immediately resuspended in 1 mL of ice-cold Sorbitol. Cells were then collected by centrifugation, resuspended in 1 mL of YPD media, and allowed to recover by incubation at 30°C at 250 rpm for 3–24 hours. Cells were then collected, resuspended in 100 μL of YPD, and plated onto drug selective media at the desired concentration. Nourseothricin (GoldBio) was used at a final concentration of 300 μg/mL for antibiotic selection of the NatMX cassette. Hygromycin B (Cayman) was used at a final concentration of 500 μg/mL antibiotic selection of the HphMX cassette. Geneticin (G418, GoldBio) was used at a final concentration of 800 μg/mL for antibiotic selection of the KanMX cassette. Zeocin (Cayman) was used at a final concentration of 600 μg/mL for antibiotic selection of the BleMX cassette in C. glabrata and 800 μg/mL in C. auris. Colonies were streaked onto fresh plates with the desired drug, and single colonies were selected and restreaked onto fresh YPD plates. Colonies were screened via colony PCR using primers indicated in Table S2 . Three independent clones were verified by PCR and analyzed for phenotypic characterizations.
10
Yeast Strain Construction and Fermentation
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YPD medium (10 g/L Bacto Yeast Extract, 20 g/L Bacto peptone and 20 g/L dextrose) was used for yeast strain construction and shake flask fermentation. For HphMx, KanMx, ble and NatMX marker selection, 300 mg/L hygromycin B (Roche Diagnostics, Basel, Switzerland), 200 mg/L G418 sulfate (Sangon Biotech, Shanghai, China), 20 mg/L phleomycin (InvivoGen, San Diego, CA, USA) and 100 mg/L nourseothricin (Gold Biotechnology, USA) sulfate was added into YPD agar plate, respectively.
For shake flask fermentation of engineered yeast strains, individual clones of the desired stain picked from YPD agar plates were inoculated into the liquid YPD medium and cultivated at 30 °C, 250 rpm overnight. Then the seed cultures were inoculated into 10 mL of liquid YPD medium in 50 mL shake flasks with an initial OD600 of 0.05, fermentation was conducted under 30 °C, 250 rpm for 96 h. The fermentation broth containing the yeast cell and medium was extracted with equal volumes of n-butanol and used for the analysis of CK, DMG, DM and PPD.
For shake flask fermentation of engineered yeast strains, individual clones of the desired stain picked from YPD agar plates were inoculated into the liquid YPD medium and cultivated at 30 °C, 250 rpm overnight. Then the seed cultures were inoculated into 10 mL of liquid YPD medium in 50 mL shake flasks with an initial OD600 of 0.05, fermentation was conducted under 30 °C, 250 rpm for 96 h. The fermentation broth containing the yeast cell and medium was extracted with equal volumes of n-butanol and used for the analysis of CK, DMG, DM and PPD.
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