Sucrose gradient fractionation
E. coli strains from the Keio collection were streaked onto LB
agar plates and grown overnight at 37°C. A single colony of each strain
was inoculated into LB and aerated at 37°C overnight. The following
morning, the culture densities were determined via spectrophotometer
(Biochrom Ultrospec 7000); the cells were diluted to a final
OD600 of 0.05-0.06 in LB medium (150-250mL) and grown
until OD600 = 0.3-0.35. The cultures were then split
into two flasks, in which the chloramphenicol treatment was carried out,
while the other was grown as a control for 2 hours.
The cells were transferred into centrifugation bottles, cooled on ice
and pelleted at 4000xg, at +4°C for 10 minutes. The supernatant was
removed, and the cell pellet was snap-frozen in liquid nitrogen and
stored at -80°C. The cells were dissolved in 1 mL of lysis buffer
consisting of 25 mM Tris-HCl pH 7.9, 60 mM KCl, 60 mM
NH4Cl, 6 mM MgCl2, 5% glycerol
supplemented with 1mM PMSF, protease inhibitor (Roche, #04693159001)
and 5mM βME added freshly to the buffer before the lysis. The cells were
lysed using FastPrep homogenizer (MP Biomedicals) by three 40-second
pulses at 4.0 m/s, chilling on ice for 5 min between the cycles. The
beads were purchased from BioSpec Products, and 0.4 gram of 0.5mm
Zirconia/Silica beads (BioSpec, #11079105z) and 0.9 gram of 0.1mm
Zirconia/Silica beads (BioSpec, #11079101z) was used.
The lysate was clarified by centrifugation 16,100xg for 40 minutes at
4°C. Clarified lysates were treated with 50 units/mL DNase I (MN,
#740963). The lysates were loaded onto 10-30% sucrose gradients in a
buffer containing 25mM Tris-HCl pH 7.9, 100mM KCl, 10mM
MgCl2, supplemented with 5mM βME. The gradients were
centrifugated at 20,400 rpm for 17 h at 4°C in an SW-28 Beckman Coulter
rotor (ω2t=2.8e+11). The samples from the gradient
were pumped starting from the bottom through a spectrophotometer (Econo
UV Monitor, BIO-RAD), which can detect A254 as a readout. The data was
recorded by Data Acquisition software (DataQ Instruments) and imported
into R for plotting (R Core Team, 2022).