2 Materials
2.1 Cell Culture
for Purification of C+,
CÀ, and Mpy-Bound
CÀ Ribosomes from
M. smegmatis
1. A frozen stock of M. smegmatis, mc
2 155 [22].
2. 7H9ADCTw: Dissolve 4.7 g of Middlebrook 7H9 base
(BD Difco) in 890 mL of double-distilled water. Mix 2 mL of
100% glycerol and adjust the volume to 900 mL. Autoclave and
cool to room temperature. To each 90 mL aliquot of the base
medium, add 10 mL of ADC enrichment (albumin/dextrose/
catalase—BD Difco) and 250 μL of 20% v/v Tween 80 (final
concentration of 0.05% v/v).
3. 40 mM TPEN (N,N,N
0 ,N
0 -Tetrakis(2-pyridylmethyl)ethylenediamine): Dissolve 100 mg of TPEN (Sigma) in 4 mL ethanol.
Bring the volume to 5.9 mL. Store at À20
C.
4. 1 M ZnSO 4 ∙7H 2 O: Dissolve 2.875 g of zinc sulfate heptahydrate (ZnSO 4 ∙7H 2 O) to 9 mL double-distilled water. Bring the
volume to 10 mL. Filter-sterilize the solution and store at 4
C.
5. Sauton’s medium base: Dissolve 0.5 g KH 2 PO 4 , 0.5 g MgSO 4 ,
4 g L-asparagine monohydrate, 2 g citric acid, and 0.05 g ferric
ammonium citrate to 900 mL deionized water. Add 60 mL
glycerol. Adjust pH to 7.0 with 1 M NaOH. Autoclave and
store at room temperature. If culturing planktonic cells, add
2.5 mL of sterile 20% Tween 80 per liter of medium before use.
6. Low-zinc Sauton’s medium: Add 25 μL of 40 mM TPEN stock
per liter of sterile Sauton’s medium base.
7. High-zinc Sauton’s medium: Add 1 mL of sterile 1 M
ZnSO 4 ∙7H 2 O per liter of sterile Sauton’s medium base.
8. PBSTw: Add 250 μL of 20% v/v sterile Tween 80 (final concentration of 0.05% v/v) to 100 mL of sterile phosphatebuffered saline (pH 7.4).
9. Centrifuge and rotor similar to Thermo Sorvall Legend XTR
with Thermo Scientific Swing-out rotor and corresponding
adapters and tubes.
10. Centrifuge and rotor similar to Thermo Sorvall Lynx 4000
with F14-6 Â 250y rotor.
2.2 Cell Culture
for Purification of C+
and Hibernating CÀ
Ribosomes from
M. tuberculosis
1. A frozen stock of M. tuberculosis, mc
2 7000 [23] (see Note 1).
2. 10% Pan solution (10% w/v D-Pantothenic acid hemicalcium
salt): Dissolve 1 g of patothenate in approximately 9 mL of
double-distilled water and make up the volume to 10 mL.
Filter-sterilize the solution and store at 4
C until further use
(see Note 1).
3. 7H9OADCPanTw: Dissolve 4.7 g of Middlebrook 7H9 base
(BD Difco) in 890 mL of double-distilled water. Mix 2 mL of
100% glycerol and adjust the volume to 900 mL. Autoclave and
Purification of Three Types of Ribosomes from Mycobacteria
153
2.1 Cell Culture
for Purification of C+,
CÀ, and Mpy-Bound
CÀ Ribosomes from
M. smegmatis
1. A frozen stock of M. smegmatis, mc
2 155 [22].
2. 7H9ADCTw: Dissolve 4.7 g of Middlebrook 7H9 base
(BD Difco) in 890 mL of double-distilled water. Mix 2 mL of
100% glycerol and adjust the volume to 900 mL. Autoclave and
cool to room temperature. To each 90 mL aliquot of the base
medium, add 10 mL of ADC enrichment (albumin/dextrose/
catalase—BD Difco) and 250 μL of 20% v/v Tween 80 (final
concentration of 0.05% v/v).
3. 40 mM TPEN (N,N,N
0 ,N
0 -Tetrakis(2-pyridylmethyl)ethylenediamine): Dissolve 100 mg of TPEN (Sigma) in 4 mL ethanol.
Bring the volume to 5.9 mL. Store at À20
C.
4. 1 M ZnSO 4 ∙7H 2 O: Dissolve 2.875 g of zinc sulfate heptahydrate (ZnSO 4 ∙7H 2 O) to 9 mL double-distilled water. Bring the
volume to 10 mL. Filter-sterilize the solution and store at 4
C.
5. Sauton’s medium base: Dissolve 0.5 g KH 2 PO 4 , 0.5 g MgSO 4 ,
4 g L-asparagine monohydrate, 2 g citric acid, and 0.05 g ferric
ammonium citrate to 900 mL deionized water. Add 60 mL
glycerol. Adjust pH to 7.0 with 1 M NaOH. Autoclave and
store at room temperature. If culturing planktonic cells, add
2.5 mL of sterile 20% Tween 80 per liter of medium before use.
6. Low-zinc Sauton’s medium: Add 25 μL of 40 mM TPEN stock
per liter of sterile Sauton’s medium base.
7. High-zinc Sauton’s medium: Add 1 mL of sterile 1 M
ZnSO 4 ∙7H 2 O per liter of sterile Sauton’s medium base.
8. PBSTw: Add 250 μL of 20% v/v sterile Tween 80 (final concentration of 0.05% v/v) to 100 mL of sterile phosphatebuffered saline (pH 7.4).
9. Centrifuge and rotor similar to Thermo Sorvall Legend XTR
with Thermo Scientific Swing-out rotor and corresponding
adapters and tubes.
10. Centrifuge and rotor similar to Thermo Sorvall Lynx 4000
with F14-6 Â 250y rotor.
2.2 Cell Culture
for Purification of C+
and Hibernating CÀ
Ribosomes from
M. tuberculosis
1. A frozen stock of M. tuberculosis, mc
2 7000 [23] (see Note 1).
2. 10% Pan solution (10% w/v D-Pantothenic acid hemicalcium
salt): Dissolve 1 g of patothenate in approximately 9 mL of
double-distilled water and make up the volume to 10 mL.
Filter-sterilize the solution and store at 4
C until further use
(see Note 1).
3. 7H9OADCPanTw: Dissolve 4.7 g of Middlebrook 7H9 base
(BD Difco) in 890 mL of double-distilled water. Mix 2 mL of
100% glycerol and adjust the volume to 900 mL. Autoclave and
Purification of Three Types of Ribosomes from Mycobacteria
153