ligase, shrimp alkaline phosphatase, materials for DNA amplification via polymerase chain reaction, DNA/plasmid extraction,
and DNA gel electrophoresis materials).
5. Zero Blunt TOPO PCR Cloning kit for sequencing (ThermoFisher Scientific, Waltham, MA) (see Note 1).
6. E. coli TOP10 cells.
7. Appropriate M. tuberculosis strain. We routinely use
M. tuberculosis CDC1551 and Erdman strains (Erdman is
used in mice infection experiments) (see Note 2).
8. T-25 flasks with filter caps for routine growth of M. tuberculosis.
2.2 Construction of
Dual Fluorescent
Reporter M.
tuberculosis Strains
1. See items 1–8 under Subheading 2.1.
2. Appropriate cloning vector. Used in particular here is pCherry3
(containing mCherry driven by the smyc promoter) [33].
2.3 Characterization
of Fluorescent
Reporter M.
tuberculosis Strains
1. Reporter M. tuberculosis strains (see Subheadings 2.1 and 2.2).
2. 7H9, pH 7.0: Middlebrook 7H9 broth supplemented with
0.2% v/v glycerol and 0.05% v/v Tween 80, pH 7.0 (buffered
with 100 mM 3-(N-Morpholino)-propanesulfonic acid).
3. 7H9, pH 5.7: Middlebrook 7H9 broth supplemented with
0.2% v/v glycerol and 0.05% v/v Tween 80, pH 5.7 (buffered
with 100 mM 2-(N-Morpholino) ethanesulfonic acid,
monohydrate).
4. 7H9, pH 7.0, 250 mM NaCl: Middlebrook 7H9 broth supplemented with 0.2% v/v glycerol and 0.05% v/v Tween
80, pH 7.0, with 250 mM NaCl (buffered with 100 mM
3-(N-Morpholino)-propanesulfonic acid).
5. 7H9, pH 5.7, 250 mM NaCl: Middlebrook 7H9 broth supplemented with 0.2% v/v glycerol and 0.05% v/v Tween
80, pH 5.7, with 250 mM NaCl (buffered with 100 mM
2-(N-Morpholino) ethanesulfonic acid, monohydrate).
6. pH standards 4.0, 7.0, and 10.0 (see Note 3).
7. 10 M sodium hydroxide (NaOH) (see Note 4).
8. pH meter.
9. T-25 flasks with filter caps for routine growth of M. tuberculosis.
10. 125 mL disposable Erlenmeyer flasks with filter caps for hypoxia or nitric oxide stress assays.
11. 2 mL screw cap tubes.
12. Phosphate-buffered saline (PBS), pH 7.4: 1.058 mM
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
13. 4% w/v paraformaldehyde, prepared in PBS (PFA).
14. DETA NONOate.
15. PBS-Tw (0.1%): 0.1% v/v Tween 80 in PBS, pH 7.4.
Exploiting Fluorescent Reporter M. tuberculosis Strains
367
and DNA gel electrophoresis materials).
5. Zero Blunt TOPO PCR Cloning kit for sequencing (ThermoFisher Scientific, Waltham, MA) (see Note 1).
6. E. coli TOP10 cells.
7. Appropriate M. tuberculosis strain. We routinely use
M. tuberculosis CDC1551 and Erdman strains (Erdman is
used in mice infection experiments) (see Note 2).
8. T-25 flasks with filter caps for routine growth of M. tuberculosis.
2.2 Construction of
Dual Fluorescent
Reporter M.
tuberculosis Strains
1. See items 1–8 under Subheading 2.1.
2. Appropriate cloning vector. Used in particular here is pCherry3
(containing mCherry driven by the smyc promoter) [33].
2.3 Characterization
of Fluorescent
Reporter M.
tuberculosis Strains
1. Reporter M. tuberculosis strains (see Subheadings 2.1 and 2.2).
2. 7H9, pH 7.0: Middlebrook 7H9 broth supplemented with
0.2% v/v glycerol and 0.05% v/v Tween 80, pH 7.0 (buffered
with 100 mM 3-(N-Morpholino)-propanesulfonic acid).
3. 7H9, pH 5.7: Middlebrook 7H9 broth supplemented with
0.2% v/v glycerol and 0.05% v/v Tween 80, pH 5.7 (buffered
with 100 mM 2-(N-Morpholino) ethanesulfonic acid,
monohydrate).
4. 7H9, pH 7.0, 250 mM NaCl: Middlebrook 7H9 broth supplemented with 0.2% v/v glycerol and 0.05% v/v Tween
80, pH 7.0, with 250 mM NaCl (buffered with 100 mM
3-(N-Morpholino)-propanesulfonic acid).
5. 7H9, pH 5.7, 250 mM NaCl: Middlebrook 7H9 broth supplemented with 0.2% v/v glycerol and 0.05% v/v Tween
80, pH 5.7, with 250 mM NaCl (buffered with 100 mM
2-(N-Morpholino) ethanesulfonic acid, monohydrate).
6. pH standards 4.0, 7.0, and 10.0 (see Note 3).
7. 10 M sodium hydroxide (NaOH) (see Note 4).
8. pH meter.
9. T-25 flasks with filter caps for routine growth of M. tuberculosis.
10. 125 mL disposable Erlenmeyer flasks with filter caps for hypoxia or nitric oxide stress assays.
11. 2 mL screw cap tubes.
12. Phosphate-buffered saline (PBS), pH 7.4: 1.058 mM
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
13. 4% w/v paraformaldehyde, prepared in PBS (PFA).
14. DETA NONOate.
15. PBS-Tw (0.1%): 0.1% v/v Tween 80 in PBS, pH 7.4.
Exploiting Fluorescent Reporter M. tuberculosis Strains
367