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.
Hygromycin b
Manufactured by Cayman Chemical
Sourced in United States
Hygromycin B is a laboratory-grade antibiotic used in scientific research. It inhibits protein synthesis in both prokaryotic and eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Nourseothricin, by GoldBio (2 mentions)
Gene pulser cuvette, by Bio-Rad (2 mentions)
Geneticin g418, by GoldBio (2 mentions)
Zeocin, by Cayman Chemical (2 mentions)
Eporator, by Eppendorf (2 mentions)
Cycloheximide (chx), by Cayman Chemical (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Hct116, by American Type Culture Collection (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Doxycycline, by Cayman Chemical (1 mentions)
Viafect, by Promega (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Antibodies to β tubulin, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Gemini Bio (1 mentions)
Streptomycin, by Gemini Bio (1 mentions)
Apramycin, by Merck Group (1 mentions)
Retapamulin, by Merck Group (1 mentions)
Eravacycline, by MedChemExpress (1 mentions)
Streptomycin, by Santa Cruz Biotechnology (1 mentions)
6 protocols using hygromycin b
1
CRISPR-Mediated Gene Editing in Yeast
2
Cellular Responses to Oxidative Stress
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Hygromycin B and doxycycline were purchased from Cayman (Ann Arbor, MI, USA). N-acetyl-l -cysteine (NAC) and antibodies to β-tubulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). HIF- inhibitor (sc-205346) and antibodies to HIF-1α (sc-10790) or Nrf2 (sc-365949) were obtained from Santa Cruz Biotechnology (CA, USA). Antibodies to CXCR4 (35-8800) were obtained from Invitrogen, Thermo Fisher Scientific. (Waltham, MA, USA). 2′,7′–dichlorofluorescin diacetate (DCF-DA) was purchased from Molecular Probe (Eugene, OR, USA). CellTiter-Glo substrates and ViaFect™ were purchased from Promega Co. (Madison, WI, USA). pCDNA3.1-Nrf2 plasmids were kindly provided from Professor Byung-Chul Kim, Division of Life Sciences, Kangwon National University, Chuncheon, Republic of Korea. Except indicated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA)41 (link).
3
Culturing Human Cell Lines for Experimentation
4
CRISPR-based Gene Editing in Candida
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Twenty microliters of prepared cells, 1–3 µg of drug resistance cassette DNA, CRISPR mix, and RNAse-free water to a final volume of 45 µL was mixed and transferred to a Bio-Rad Gene Pulser cuvette (0.2-cm gap). Cells were pulsed using an Eppendorf Eporator at 1,500 V and immediately resuspended in 1 mL of ice-cold sorbitol. Cells were 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. Recovered cells were resuspended in 100 µL of YPD and plated onto drug-selective media. 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. Geneticin (G418, GoldBio) was used at a final concentration of 800 µg/mL. Zeocin (Cayman) was used at a final concentration of 600 µg/mL for C. glabrata and 800 µg/mL for C. auris. For double selection for C. albicans using SAT1 and BleMX or KanMX, plates contained 200 µg/mL Nourseothricin and 800 µg/mL Zeocin or 800 µg/mL G418 sulfate. Colonies were screened via PCR using primers indicated in Table S2. Three independent clones were used for phenotypic characterizations.
5
Reconstitution of E. coli 70S Ribosomes
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In vitro reconstituted E. coli 70S ribosomes were generated from the E. coli K12 strain BW25113, as described previously56 (link). Antibiotic–ribosome samples were generated by incubating antibiotic cocktails 1–5 with E. coli 70S ribosomes in buffer A (50 mM HEPES-KOH, pH 7.5, 25 mM Mg(OAc)2, 80 mM NH4Cl, 100 mM KOAc, 1 mM DTT, 0.05% DDM) at 37 °C for 15 min, before being frozen at −80 °C until use. Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).
6
Cell Culture Protocols for Cancer Research
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Human colorectal cancer line HCT116 and HEK-293 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). 293pTP and RAPA cells derived from HEK-293 cells as previously characterized.40 (link), 41 (link) All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in 5% CO2 as described.42 (link), 43 , 44 (link) Cycloheximide (CHX) and hygromycin B were purchased from Cayman Chemical (Ann Arbor, MI). Unless indicated otherwise, other reagents were purchased from Sigma–Aldrich (St. Louis, MO) or Thermo Fisher Scientific (Waltham, MA).
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