13. 50 mg/mL Gentamicin (5000Â): Make a stock solution in
distilled water. Filter sterilize and store at À20
C. Use at a
working concentration of 10μg/mL.
2.1.2 Construction
of Native Promoter-Driven
Complementing Vector
Using pUC-Gm-Int.
1. pUC-Gm-Int (available from Tanya Parish:Tanya.Parish@seattlechildrens.org).
2. Mycobacterial genomic DNA.
3. Primers designed to amplify the gene being complemented.
4. PCR reagents, thermocycler, and consumables.
5. PCR cleanup or gel extraction Kit, e.g., QiaQuick, Qiagen, or
similar.
6. Agarose gel electrophoresis equipment.
7. T-A cloning vector, pSC-A (Agilent), or similar.
8. Competent E. coli strains suitable for recombinant genetic
work, e.g., DH5α or XL1.
9. Plasmid purification kit (Qiagen or similar).
10. See items 11–13 under Subheading 2.1.1.
11. LA-Gm: To LB media add 15 g of Bacto Agar before autoclaving. Make up to 1 L with distilled water. Autoclave to sterilize.
Allow to cool, add 10μg/mL gentamycin, mix well and pour
onto petri dishes.
2.2 Obtaining
a Delinquent Strain
(Δ
0 int)
1. M. tuberculosis SCO culture (see Note 1).
2. 490 cm
3 roller bottles (Corning).
3. Roller incubator (set at 37
C).
4. 2 M glycine. Filter sterilize. Store at 4
C.
5. Oleic albumin dextrose catalase (OADC) supplement (Becton
Dickinson). Store at 4
C and use at a final concentration of
10% v/v.
6. Glycerol. Make a 10% v/v stock in water and filter sterilize.
Store at 4
C.
7. 7H9-Tw medium: Dissolve 4.7 g of Middlebrook 7H9 broth
base (Difco) in 900 mL deionized water. Add 5 mL of 20%
(v/v) Tween 80 (final concentration of 0.05% v/v). Mix well
and filter sterilize or autoclave. Add 100 mL of OADC immediately before use.
8. 7H9-Tw-KH medium (see Note 1): Dissolve 4.7 g of Middlebrook 7H9 broth base (Difco) in 900 mL deionized water.
Add 5 mL of 20% (v/v) Tween 80 (final concentration of
0.05% v/v). Mix well and filter sterilize or autoclave. Add
100 mL of OADC and kanamycin stock to give final concentration of 20μg/mL, and hygromycin to give final concentration of 50μg/mL, immediately before use.
Gene Switching and Essentiality Testing
291
distilled water. Filter sterilize and store at À20
C. Use at a
working concentration of 10μg/mL.
2.1.2 Construction
of Native Promoter-Driven
Complementing Vector
Using pUC-Gm-Int.
1. pUC-Gm-Int (available from Tanya Parish:Tanya.Parish@seattlechildrens.org).
2. Mycobacterial genomic DNA.
3. Primers designed to amplify the gene being complemented.
4. PCR reagents, thermocycler, and consumables.
5. PCR cleanup or gel extraction Kit, e.g., QiaQuick, Qiagen, or
similar.
6. Agarose gel electrophoresis equipment.
7. T-A cloning vector, pSC-A (Agilent), or similar.
8. Competent E. coli strains suitable for recombinant genetic
work, e.g., DH5α or XL1.
9. Plasmid purification kit (Qiagen or similar).
10. See items 11–13 under Subheading 2.1.1.
11. LA-Gm: To LB media add 15 g of Bacto Agar before autoclaving. Make up to 1 L with distilled water. Autoclave to sterilize.
Allow to cool, add 10μg/mL gentamycin, mix well and pour
onto petri dishes.
2.2 Obtaining
a Delinquent Strain
(Δ
0 int)
1. M. tuberculosis SCO culture (see Note 1).
2. 490 cm
3 roller bottles (Corning).
3. Roller incubator (set at 37
C).
4. 2 M glycine. Filter sterilize. Store at 4
C.
5. Oleic albumin dextrose catalase (OADC) supplement (Becton
Dickinson). Store at 4
C and use at a final concentration of
10% v/v.
6. Glycerol. Make a 10% v/v stock in water and filter sterilize.
Store at 4
C.
7. 7H9-Tw medium: Dissolve 4.7 g of Middlebrook 7H9 broth
base (Difco) in 900 mL deionized water. Add 5 mL of 20%
(v/v) Tween 80 (final concentration of 0.05% v/v). Mix well
and filter sterilize or autoclave. Add 100 mL of OADC immediately before use.
8. 7H9-Tw-KH medium (see Note 1): Dissolve 4.7 g of Middlebrook 7H9 broth base (Difco) in 900 mL deionized water.
Add 5 mL of 20% (v/v) Tween 80 (final concentration of
0.05% v/v). Mix well and filter sterilize or autoclave. Add
100 mL of OADC and kanamycin stock to give final concentration of 20μg/mL, and hygromycin to give final concentration of 50μg/mL, immediately before use.
Gene Switching and Essentiality Testing
291