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. For cell lysis, the frozen cell pellets were thawed on
ice and then taken up 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. 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) on ice and centrifuged for 10 minutes as above. The
AU260 was monitored, and 50U was loaded. The lysates
were loaded onto 10-30% sucrose gradients in a buffer containing 25 mM
Tris-HCl pH 7.9, 100 mM KCl, 10 mM 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 from the bottom through a
spectrophotometer (Econo UV Monitor, BIO-RAD), which can detect
absorbance at 254 nm as a readout. The data was recorded by Data
Acquisition software (DataQ Instruments) and imported into R for
plotting (R Core Team, 2022).