M. abscessus CIP104536T (S and R variants), M. massiliense CIP108297T (S and R variants) and the clinical strains M. massiliense GD01 (rough) and M. abscessus T56 (rough) were routinely grown and maintained at 37°C in Middlebrook 7H9 broth (BD Difco) supplemented with 10% oleic acid, albumin, dextrose, catalase (OADC; BD Difco) and 0.025% Tyloxapol (Sigma-Aldrich) (7H9OADC/T), or on Middlebrook 7H10 supplemented with 10% OADC enrichment (7H10OADC). Fluorescent GD01 was generated using the pTEC27 expressing tdTomato (Takaki et al., 2013 (link)). Red fluorescent colonies were selected on 7H10OADC/T supplemented with 1000 µg/ml Hygromycin (Euromedex) and maintained in 7H9OADC/T supplemented with 500 µg/ml Hygromycin. Fluorescent M. abscessus CIP104536T and M. massiliense CIP108297T strains have previously been described (Bernut et al., 2014a (link)). M. smegmatis mc2155 was grown at 37°C in Middlebrook 7H9 broth as previously described (Jacobs-Sera et al., 2012 (link)).
Hygromycin
Manufactured by Euromedex
Hygromycin is a laboratory reagent used in cell and molecular biology. It functions as an antibiotic that inhibits protein synthesis in eukaryotic cells and is commonly used for selection of transformed cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Tyloxapol, by Merck Group (1 mentions)
Ez yeast transformation kit, by MP Biomedicals (1 mentions)
Kanamycin, by Euromedex (1 mentions)
Tween 80, by Merck Group (1 mentions)
7 protocols using hygromycin
1
Cultivation and Genetic Manipulation of Mycobacterial Strains
2
Cell Culture and Stable Cell Lines
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All U2OS (American Type Culture Collection ATCC HTB-96, human osteosarcoma, female) and NIH/3T3 (ATCC CRL-1658, mouse embryonic fibroblast, male) cell lines were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (EUROBIO), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen) and the appropriate selection antibiotics.
| Stable cell line (reference if not generated in this study) | Selection antibiotics |
|---|---|
| U2OS H2A.X-SNAP WT form | G418 |
| U2OS H2A.X-SNAP S139A mutant | G418 |
| U2OS H2A.X-SNAP S139E mutant | G418 |
| U2OS H2A.Z.1-SNAP | G418 |
| U2OS H2A.Z.2-SNAP | G418 |
| U2OS H2A-SNAP | G418 |
| U2OS H3.3-SNAP (Dunleavy et al., 2011 (link)) | G418 |
| NIH/3T3 H2A.X-SNAP + GFP-DDB2 | G418 + Hygromycin |
3
Screening for Yeast Offspring Viability
4
Yeast Strain Maintenance and Sporulation
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Standard media conditions were used (YPD 1% yeast extract, 2% peptone and 2% glucose at 30 °C) for growth and maintenance of the strains. A final concentration of 200 μg ml−1 hygromycin (Euromedex) was supplemented to maintain the plasmid CEN_SUP7 or CEN_Ctrl carrying a resistance marker gene hygroMX. Sporulation was induced on potassium acetate plates (1% potassium acetate, 2% agar). The 5-FOA selection plate was made by supplementing 25 μg ml−1 uracil and 1 mg ml−1 5-FOA in a synthetic complete uracil dropout media (SC-URA). Detailed media composition for the screening of offspring viability and stress conditions are listed in Supplementary Table 2 . All procedures are performed at 30 °C unless otherwise indicated.
5
Yeast Segregant Panel Phenotyping
6
Transposition of Ty1 Elements in Yeast
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Yeast cells were grown on YPD (yeast extract 1%, peptone 2%, dextrose 2%) at 30 °C. When required, the temperature was adjusted to 34 °C or to 22 °C that are respectively restrictive or permissive to Ty1 transposition (Paquin and Williamson 1986 (link); Lawler et al. 2002 (link)). Geneticin (Euromedex) to a final concentration of 200 µg/ml was added to obtain the selective YPD-G418 media. The solid selective media YPD-hygro was supplemented with hygromycin (Euromedex) to a final concentration of 200 µg/ml and with 2% agar (Bacto agar Difco). The composition of the nitrogen-depleted media was as follows: 0.67% Yeast Nitrogen Base without amino acids and ammonium sulfate (MP Biomedicals), 2% D-glucose, 0.05 mM ammonium sulfate. Yeast strains were transformed by the pCeTyX plasmid carrying Ty1hygro-AI with the EZ-Yeast Transformation Kit (MP Biomedicals).
7
Mycobacterium Growth and Culturing Protocol
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The M. canettii, MTB and M. smegmatis strains used in this study are listed in Supplementary Table 3. All strains were cultured in Middlebrook 7H9 broth (BD Difco) supplemented with 0.2% glycerol (w/v) and 10% Middlebrook albumin-dextrose-catalase enrichments (ADC; BD BBL), with 0.05% Tween 80 (Sigma) (7H9/ADC/Tween) unless otherwise described. To grow recombinant strains, the antibiotics hygromycin (Euromedex) and kanamycin (Euromedex) were added as required at final concentrations of 50 µg/ml and 40 µg/ml respectively. Cultures were agitated approximately twice per week and maintained for 3-4 weeks at 37ᵒC before use. During the experimental evolution cycles, the antibiotic mixture PANTA (BD BBL) was added at a dilution of 25 µl for 10 ml 7H9/ADC/Tween for in vitro cultures. For growth on solid medium, 7H11 agar base (BD BBL) was supplemented with 0.5% glycerol and 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC; BD BBL). Counting of CFUs on 7H11 agar plates was performed after 2, 3 and 4 weeks of incubation at 37ᵒC.
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