Mbr 022up

To examine effects of drugs on the abnormal oligomerization of SOD1 in vitro, apo-SOD1(G37R)^S-S (20 μM, 150 μL) in a buffer at pH 7.4 containing 100 mM Na-Pi, 100 mM NaCl and 5 mM EDTA (NNE buffer) with 1 M Gdn was first prepared in each well of a 96-well-plate. Then, 1.5 μL of a 2.0 g/L stock solution of drugs in DMSO was added to the protein sample solution in a well of the plate (the final concentration of drugs tested was 20 μg/mL). In this study, we have examined 640 drugs in the FDA approved drug library (#BML-2841J-0100, Japanese version, Enzo Life Science). The oligomerization reaction was started by shaking the samples with a POM ball (3/32 inch, SANPLATEC) in a plate shaker (MBR-022UP, TAITEC) at 1200 rpm, 37°C and monitored by the increase of solution turbidity (absorbance increase at 350 nm). After 12 h of agitation, the samples were collected and ultracentrifuged at 110,000 × g for 15 min. to obtain soluble supernatant and insoluble pellet separately. After removing Gdn in the soluble supernatant with PAGE Clean Up Kit (nacalai tesque), the proteins in both fractions were analyzed by non-reducing SDS-PAGE.

The E. coli cells were inoculated from glycerol stocks into test tubes containing 3 mL of either 3.5 mM OAV-supplemented or 10.5 mM glucose-supplemented minimal medium and incubated in a bioshaker (MBR-022UP, Taitec, Japan) at 200 rpm and 37 °C as precultures. The precultures were subsequently transferred to 3 mL of fresh minimal medium that was supplied with 0.035, 0.07, 0.35, 0.7, or 3.5 mM OAVs or 0.105, 0.21, 1.05, 2.1, or 10.5 mM glucose, respectively, at a common dilution rate of 1000-fold. Every 10 test tubes of parallel cultures were applied for each concentration (a total of 10 concentrations). The changes in cell concentration, morphology, fluorescence, etc., were evaluated with an imaging flow cytometer at intervals of several hours until the cell culture reached the stationary phase. The utilization capacity was defined as the mean values (N > 5) of the maximal cell concentration (cells/mL), which were calculated according to the temporal measurements, divided by the concentrations (mM) of the supplied carbon source, i.e., OAVs or glucose. The steady population densities were measured, and the cell concentrations per mM carbon source were calculated as the population capacity. The results were summarized in Supplementary Data 6.

The E. coli strain was initially inoculated into 200 μL of the minimal medium with 3.5 mM OAVs in a flat-bottom 96 microwell plate (Corning, USA) sealed with CyclerSeal Sealing Film (Axygen, USA). The microplates were incubated in a bioshaker (MBR-022UP, Taitec, Japan) with a rotation rate of 200 rpm at 37 °C. Cells passaged several times were subjected to single colony isolation by plating the cell culture on LB agar plates (Supplementary Fig. 1A). Only one of the single colonies was selected as the Ori for the experimental evolution. The selected colony was inoculated in 3 mL of the minimal medium with 3.5 mM OAVs and grown until approximately 10⁸ cells/mL. The resultant cell culture was preserved as a stock and used as the common Ori of the experimental evolution. Note that a single-nucleotide insertion of T in ygiM, encoding the antitoxin higA, was initially detected in the Ori in comparison to the E. coli strain.

PECs (5 mL) were placed into a sample vial and its pH was adjusted to 2.0 using HCl (0.1 N) to mimic the acidic condition in the stomach. Afterward, the PEC solution was incubated at 37 °C for 2 h in a shaker (M.BR-022UP, Taitec, Tokyo, Japan). Next, the pH of the PEC solution was adjusted to 8.0 using NaOH (0.5 N) to simulate the alkaline environment in the small intestine. The adjusted PEC solution was placed at 37 °C for 4 h in a shaker. Particle size and surface zeta potential were measured by DLS at every hour.

The E. coli cells were inoculated from glycerol stocks into test tubes containing 3 mL of 10.5 mM glucose-supplemented minimal medium and incubated in a bioshaker (MBR-022UP, Taitec, Japan) at 200 rpm and 37 °C as precultures. The precultures were diluted 1000-fold with fresh minimal medium supplied with 0.105, 0.21, 1.05, 2.1, or 10.5 mM glucose and subsequently loaded into flat-bottom 96-well microplates (Corning, USA) in six wells with locations varied per culture condition, as described previously⁷⁷ . The microplates were incubated in a plate reader (Synergy H1, BioTek, USA) with continuous orbital shaking at 282 cpm and 37 °C. Growth was monitored by measuring the absorbance at 600 nm, and readings were obtained at 30 min intervals for 20–30 h. The growth rate was calculated according to the changes in OD₆₀₀, as described previously⁸¹ .

Prior to the experiment, the PrismaP HT buffer was filtered through a 0.45-mm syringe filter (GL Sciences Inc., Tokyo, Japan). A sample of the solid dispersions (5 mg) corresponding to 1 mg of IMC was added to 2 mL of the filtered PrismaP HT Buffer. They were shaken at 500 rpm at 37 °C for 4 h using a well plate shaker, M BR-022UP (TAITEC Corporation, Saitama, Japan), followed by filtration through a 0.45-μm syringe filter (GL Sciences Inc., Tokyo, Japan). Approximately 100 μL of the filtrate sample was added to a cuvette to measure dynamic light scattering (DLS) using Zetasizer Nano-ZS (Malvern Panalytical Ltd., Malvern, UK). The mean diameter was calculated using photon correlation spectroscopy, and the attenuation and measurement settings were optimized automatically by Zetasizer Software version 8.01 (Malvern Panalytical Ltd., Malvern, UK).