Amicon ultrafiltration system

Streptococcus mutans MVs were isolated as described by Liao et al. (2014) (link), with some modifications. Briefly, S. mutans strains were grown in 500 mL of BHI medium at 37°C for 16 h. Following centrifugation for 15 min at 4°C at 6,000 × g to remove cells, the cell-free culture supernatants were spun for 15 min at 4°C at 10,000 × g to remove cell debris. The resulting supernatants were filtered through 0.22-μm filters (Millipore, MMAS, United States) and then concentrated using a 100 kDa Amicon ultrafiltration system (Millipore, MMAS, United States). Subsequently, the concentrates were centrifuged at 100,000 × g for 70 min at 4°C and the pellets were washed once with sterile PBS before being resuspended in sterile PBS.
The MV yield was calculated by measuring protein concentration using a BCA assay (CWBIO, Beijing, China). For morphological analysis, TEM (H7650, Hitachi, Japan) was used to observe and identify the presence of MVs (Vargas et al., 2015 (link)). A 10 μL suspension of MVs was adhered to formvar/carbon-coated nickel TEM grids, negatively stained for 1 min with 3% uranyl acetate, washed with ddH₂O and observed by TEM at an acceleration voltage of 80 kV. The size distribution and diameter of MVs were measured by NTA (NanoSight NS300, Malvern, United Kingdom), as previously described (Arab et al., 2019 (link)).

The crude enzymatic extracts produced by C. cubensis and com- mercial cellulase (Multifect® CL) were applied in a biomass saccharification experiment. The C. cubensis enzymatic extract were concentrated 5-fold before the experiment using an Amicon Ultra- filtration system (Millipore Co. – Billerica, MA, USA) and an YM-10 (Cut-off Mr 10,000 Da) membrane filter. Enzymatic saccharification of alkali-treated sugarcane bagasse was performed in 2 mL sample tubes at an initial solid concentration of 2% dry matter (w/v) in 1.5 mL of 50 mM sodium acetate buffer at pH 4.5. Enzyme loading was specified as 10 FPase units per gram of biomass with the addition of sodium azide (10 mM) and tetracycline (40 μg mL⁻¹) to the reaction mixture to inhibit microbial contamination. The reaction was carried out in an orbital shaker at 250 rpm and 50 °C for different time intervals up to 72 h. These samples were immediately heated to 100 °C to denature the enzymes, cooled and then centrifuged for 5 min at 15,000 g. Products of the saccharification assays were analyzed by high performance liquid chromatography (HPLC) with a Shimadzu series 10 A chromatograph. The HPLC was equipped with an Aminex HPX-87P column (300 × 7.8 mm) and refractive index detectors. The column was eluted with water at a flow rate of 0.6 mL min⁻¹ and 80 °C.

The gene fragment encoding His tag, G4S, myc tag, Z_EGFR:1907 affibody, G4S, and GALA peptide was PCR-amplified using affibody forward (5-GAG​GTA​CCG​GAA​GTC​GGG​GGC​GG -3′) and affibody reverse (5′-CTC​TAG​ATT​ATT​TAG​GAG​CCT​GTG​CAT​C-3′) primers using pMD18-T vector as a template. The amplified sequence was then cloned between KpnI and XbaI sites of the pET28a expression vector and the final construct was transformed and expressed in E. coli BL21 (DE3) strain. To express the recombinant protein, a 500 ml of bacterial culture supplemented with kanamycin was set and the protein expression was induced by adding IPTG (1 mM) at 37°C for 4 h. The bacterial pellet was then collected and lysed using lysis buffer (300 mM NaCl, 50 mM NaH₂PO₄, and 10 mM imidazole; pH8.0) and sonication (12 cycles of 20 s ON/OFF). Following centrifugation at 5,000 × g for 20 min, the supernatant was loaded on the Ni-NTA agarose column (QIAGEN; Hilden, Germany) equilibrated with the same lysis buffer. After washing, the recombinant protein was eluted using the elution buffer (300 mM NaCl, 50 mM NaH₂PO₄, and 250 mM imidazole; pH8.0) and finally, imidazole was removed from the protein solution and replaced with PBS using an Amicon ultrafiltration system (3 kDa cutoff filter) (Millipore, Billerica, MA, United States). The concentration of the purified fusion protein was determined by the Bradford assay.

EVs were purified from S. aureus culture supernatants as described previously¹⁸ . Briefly, sub-cultured cells at the end of the exponential phase were diluted in 1 L of fresh BHI medium. The cultures were grown until the early stationary phase to optimise the number of EVs that could be recovered. The cells were then pelleted at 6,000 g for 15 min and the supernatant fraction was filtered through a 0.22 µm vacuum filter (PES). The filtrate was concentrated using the Amicon ultrafiltration system (Millipore) with a 100 kDa filter and subjected to ultracentrifugation at 150,000 g for 120 min at 4 °C. The pellet was then re-suspended in 8% sucrose in tris-buffered saline (TBS) (150 mM NaCl, 50 mM Tris-Cl, pH 7.5), overlaid with sucrose dilutions ranging from 8% to 68% in TBS and centrifuged at 100,000 g for 150 min at 4 °C in a SW 55 Ti rotor (Beckman Coulter). The different density fractions were collected, and those containing a similar number of EVs, with a similar particle-size distribution and protein pattern were pooled, centrifuged at 150,000 g for 120 min at 4 °C and re-suspended in TBS. EV fractions and isolated EV samples were routinely verified by electron microscopy and Bradford assay (Bio-Rad) and quantified using Nanoparticle Tracking Analysis before being stored at −20 °C until use.

EVs were purified from S. aureus N305 culture supernatants using a method adapted from (Gurung et al., 2011 (link)). Sub-cultured cells at the end of exponential phase were diluted 1:1,000 in 1 L of fresh BHI medium and were grown until the stationary phase. After the cells were pelleted at 6,000 g for 15 min, the supernatant fraction was filtered through a 0.22 μm vacuum filter (PES) and the filtrate was concentrated around 100-fold using Amicon ultrafiltration system (Millipore) with 100 kDa filter. The resulting filtrate was subjected to ultracentrifugation at 150,000 g for 120 min at 4°C and were applied to a discontinuous sucrose density gradient (8–68%). After centrifugation at 100,000 g for 150 min at 4°C, each fraction of the gradient was collected. The fractions with density around 1.08–1.13 g/cm³ were then recovered by sedimentation at 150,000 g for 120 min and suspended in Tris-Buffered Saline (TBS) (150 mM NaCl; 50 mM Tris-Cl, pH 7.5). Purified EVs were checked for absence of bacterial contamination and stored at −20°C before use. The EVs amount were measured based on protein concentration using the Bradford reagent (Bio-Rad) and visualized by SDS-PAGE. Hereafter, the S. aureus-secreted vesicle dose correspond to the quantity of S. aureus-secreted vesicle proteins.