8. As a control, set up parallel biofilm cultures in high-zinc Sauton’s medium in exact same conditions to obtain C+ ribosomes
(see Note 8).
3.3 Cell Lysis
1. Thaw frozen cells (from step 4 under Subheading 3.1 or step 7
under Subheading 3.2) on ice and remove all residual liquid.
2. In another dry ice bucket pour liquid nitrogen to fill up about
one-fourth of the depth. Immerse both grinding jars containing their balls. Power on the Mixer Mill.
3. Add 250 μL of ice-cold low-salt HMA buffer to sample, mix it
thoroughly, and immerse in liquid nitrogen until the sample is
completely frozen.
4. Transfer the frozen cells evenly in the two prechilled grinding
jars using prechilled spatulas.
5. Mill the samples at 15 Hz for 3 min. Place the jars using a pair
of tongs in liquid nitrogen for 30 s, and mill it further for
3 min. Repeat this at least five additional cycles for M. smegmatis
cells, or seven additional cycles for M. tuberculosis cells.
6. After the final cycle of milling, place the jars in liquid nitrogen
for 30 s. The frozen powder should look white. If necessary,
repeat the milling two or three additional cycles until the white
appearance develops. Transfer all frozen powder of lysed cells
from the milling jar to 50 mL conical tubes filled with 25 mL
liquid nitrogen using a prechilled spatula. Close the conical
tubes with perforated caps (see Note 9), and place on ice.
7. Wash the grinding jars and balls in warm water and air-dry
them completely. The washed jars and balls can be reused for
additional samples by repeating steps 1–6.
8. Lysed, frozen samples can be stored at À80
C.
3.4 Purification
of Crude Ribosomes
1. Thaw lysed cells on ice. Add 10 mL of ice-cold low-salt HMA
buffer.
2. Mix thoroughly and transfer to a 50 mL centrifuge tube.
3. Wash the residual material from the conical tube twice with
5 mL ice-cold low-salt HMA buffer.
4. Centrifuge the pooled lysate for 30 min at 15,900 rpm (30,000
 g) at 4
C using Thermo Sorvall Lynx 4000 or similar
centrifuge with F21-8 x 50y or similar rotor.
5. Transfer the lysate into a prechilled ultracentrifuge tube for
TY70Ti or similar rotor. Centrifuge the samples at
42,800 rpm (~188,000 Â g, r max ) in an ultracentrifuge similar
to Beckman Coulter Optima L-90K for 2 h and 15 min at 4
C.
6. Slowly discard the supernatant, add 1 mL of ice-cold low-salt
HMA buffer to the pellet, and soak it overnight on ice.
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Yunlong Li et al.
(see Note 8).
3.3 Cell Lysis
1. Thaw frozen cells (from step 4 under Subheading 3.1 or step 7
under Subheading 3.2) on ice and remove all residual liquid.
2. In another dry ice bucket pour liquid nitrogen to fill up about
one-fourth of the depth. Immerse both grinding jars containing their balls. Power on the Mixer Mill.
3. Add 250 μL of ice-cold low-salt HMA buffer to sample, mix it
thoroughly, and immerse in liquid nitrogen until the sample is
completely frozen.
4. Transfer the frozen cells evenly in the two prechilled grinding
jars using prechilled spatulas.
5. Mill the samples at 15 Hz for 3 min. Place the jars using a pair
of tongs in liquid nitrogen for 30 s, and mill it further for
3 min. Repeat this at least five additional cycles for M. smegmatis
cells, or seven additional cycles for M. tuberculosis cells.
6. After the final cycle of milling, place the jars in liquid nitrogen
for 30 s. The frozen powder should look white. If necessary,
repeat the milling two or three additional cycles until the white
appearance develops. Transfer all frozen powder of lysed cells
from the milling jar to 50 mL conical tubes filled with 25 mL
liquid nitrogen using a prechilled spatula. Close the conical
tubes with perforated caps (see Note 9), and place on ice.
7. Wash the grinding jars and balls in warm water and air-dry
them completely. The washed jars and balls can be reused for
additional samples by repeating steps 1–6.
8. Lysed, frozen samples can be stored at À80
C.
3.4 Purification
of Crude Ribosomes
1. Thaw lysed cells on ice. Add 10 mL of ice-cold low-salt HMA
buffer.
2. Mix thoroughly and transfer to a 50 mL centrifuge tube.
3. Wash the residual material from the conical tube twice with
5 mL ice-cold low-salt HMA buffer.
4. Centrifuge the pooled lysate for 30 min at 15,900 rpm (30,000
 g) at 4
C using Thermo Sorvall Lynx 4000 or similar
centrifuge with F21-8 x 50y or similar rotor.
5. Transfer the lysate into a prechilled ultracentrifuge tube for
TY70Ti or similar rotor. Centrifuge the samples at
42,800 rpm (~188,000 Â g, r max ) in an ultracentrifuge similar
to Beckman Coulter Optima L-90K for 2 h and 15 min at 4
C.
6. Slowly discard the supernatant, add 1 mL of ice-cold low-salt
HMA buffer to the pellet, and soak it overnight on ice.
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Yunlong Li et al.