Pes membrane filter

ASC-exosomes were isolated from the ASC CM using tangential flow filtration (TFF)-based ExoSCRT technology as previously described [18 (link), 28 (link)]. During the isolation process, the excipient EDB1 generated by ExoCoBio, Inc., was added for the uniformity of exosome particle size. The CM was filtered by a polyethersulfone (PES) membrane filter (0.22 μm, Merck Millipore, Billerica, MA, USA) to remove contaminating particles, such as cell debris, microvesicles, and apoptotic bodies. The CM was concentrated by a tangential flow filtration membrane cartridge (GE Healthcare, Chicago, IL, USA) with a molecular weight cut-off of 500 kDa, and then buffer exchange to DPBS was carried out by diafiltration. The amount of protein in the isolated ASC-exosomes was approximately 0.5% of the amount in the CM. Isolated ASC-exosomes were stored at -80°C. The frozen ASC-exosomes underwent a single free-thaw cycle before use, and the characterization and profile analysis were performed according to the Minimal Information for Studies of Extracellular Vesicles 2018 [32 (link)].

For comparison of sugar consumption rate between specialist strains, engineered strains inoculated from glycerol stock were cultured in 5 mL YP media containing 20 g L⁻¹ glucose or xylose at 1% (Vol Vol⁻¹) final inoculation concentration at 30  ^°C, 250 rpm for 48 h. In the main culture, each strain was cultured in YP media containing glucose or xylose or a mixture of glucose and xylose at a final concentration of OD₆₀₀ 1 at 30 °C, 250 rpm. For yeast cell culture for ethanol fermentation, each strain was precultured as described above. The precultured strains were inoculated in 25 mL of YP media having various glucose and xylose concentrations at a final concentration of OD₆₀₀ 1 and incubated at 100 rpm and 30 °C. A bioenergy sorghum hydrolysate produced by hydrothermal pretreatment and hydrolysis by cellulases was used for fermentation experiments. The initial pH of the hydrolysate was pH 4.8 and sodium hydroxide (Sigma-Aldrich, St. Louis, MO, USA) was added to increase the pH to 5.9. Solid residues in the hydrolysates were removed by centrifugation at 12298 g for 5 min and the supernatant was sterilized using 0.22 µm PES membrane filter (Merck Millipore, Darmstadt, Germany) before fermentation.

Phage X2 propagation was conducted according to the phage lysate method [24 ,25 (link)]. In brief, 50 μL of phage (10¹⁰ PFU/mL) was added to a 500 μL overnight bacterial culture (10⁸ CFU/mL) and was shaken at 30 °C for 6 to 8 h until the culture was cleared. After the centrifugation at 10,000× g, 4 °C for 20 min and filtration through a 0.22 μm PES membrane filter (Merck Millipore Ltd., Carrigtwohill, Ireland), the phage lysate was collected and stored at 4 °C until use. Lysis activity of phage X2 on Xoo strains was examined using spot assay by mixing 500 μL of exponential-phase Xoo cultures and 5 mL of molten NA with 0.8% (w/v) of agar and pouring the mixture into NA plates to produce double agar plates. Then, 2 μL of 10-fold serial dilution of phage was spotted onto the surface of the plates. After overnight incubation at 30 °C, the phage plaque was examined and photographed.