3.2 Cell Culture
for Purification of C+
and Hibernating CÀ
Ribosomes from
M. tuberculosis
1. Inoculate 20 mL of 7H9OADCPanTw with a frozen stock of
M. tuberculosis mc
2 7000 and incubate it standing at 37
C until
OD 600 reaches 0.4.
2. Harvest the cells by centrifugation at 4000 rpm (3500 Â g) for
10 min at room temperature using the centrifuge described in
step 2 of sect. 3.1.
3. Resuspend the cells in 20 mL of PBSTw and centrifuge at
4000 rpm (3500 Â g) for 10 min.
4. Repeat the washing (steps 2 and 3) two additional times,
before resuspending it in 20 mL detergent-free Sauton’s
medium base.
5. Inoculate 200 mL of detergent-free Sauton’s medium base (see
Note 6) with washed M. tuberculosis cells from step 4 at 1:100
dilution of cells. Dispense 4 mL of the suspension in each well
of four 12-well plates. Wrap the plates with parafilm and incubate at 37
C in humidified incubator undisturbed for 7 weeks
(see Note 7).
6. Thick pellicles are expected at the air–medium interface. Pool
the pellicles in a 50 mL polystyrene conical tube by gently
lifting the pellicles from the interface using parafilm and sliding
the biomass into the conical tube using a spatula.
7. Wash the pellicles three times in PBSTw, and freeze at À80
C
until further processing for cell lysis and ribosome purification.
Fig. 1 Growth of M. smegmatis cells in Sauton’s medium base with 1 μM TPEN.
Arrows indicate the timepoints at which saturating levels of remodeled and
hibernating ribosomes were observed
Purification of Three Types of Ribosomes from Mycobacteria
157
for Purification of C+
and Hibernating CÀ
Ribosomes from
M. tuberculosis
1. Inoculate 20 mL of 7H9OADCPanTw with a frozen stock of
M. tuberculosis mc
2 7000 and incubate it standing at 37
C until
OD 600 reaches 0.4.
2. Harvest the cells by centrifugation at 4000 rpm (3500 Â g) for
10 min at room temperature using the centrifuge described in
step 2 of sect. 3.1.
3. Resuspend the cells in 20 mL of PBSTw and centrifuge at
4000 rpm (3500 Â g) for 10 min.
4. Repeat the washing (steps 2 and 3) two additional times,
before resuspending it in 20 mL detergent-free Sauton’s
medium base.
5. Inoculate 200 mL of detergent-free Sauton’s medium base (see
Note 6) with washed M. tuberculosis cells from step 4 at 1:100
dilution of cells. Dispense 4 mL of the suspension in each well
of four 12-well plates. Wrap the plates with parafilm and incubate at 37
C in humidified incubator undisturbed for 7 weeks
(see Note 7).
6. Thick pellicles are expected at the air–medium interface. Pool
the pellicles in a 50 mL polystyrene conical tube by gently
lifting the pellicles from the interface using parafilm and sliding
the biomass into the conical tube using a spatula.
7. Wash the pellicles three times in PBSTw, and freeze at À80
C
until further processing for cell lysis and ribosome purification.
Fig. 1 Growth of M. smegmatis cells in Sauton’s medium base with 1 μM TPEN.
Arrows indicate the timepoints at which saturating levels of remodeled and
hibernating ribosomes were observed
Purification of Three Types of Ribosomes from Mycobacteria
157