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20 protocols using EmulsiFlex-B15

Various amounts of irinotecan, the lipid mixture (tricaprin and triethanolamine at the weight ratio of 4:1) and combined surfactant (Tween 80 and Span 20 at a weight ratio of 4:1) were gently mixed at 45 °C. These blends (1 g) were dispersed in 10 mL of deionised water, homogenised utilising a homogeniser (T-8-Ultra-Turrax, IKA; Königswinter, Germany) and a high-pressure homogeniser (Emulsiflex B15, Avestin; Ottawa, ON, Canada) using cycles of 500–1000 bar, and cooled down to 25 °C in order to produce the irinotecan-loaded SLN dispersions.
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XL1-Blue cell extract was created using the following protocol: XL1-Blue competent cells (Agilent Technologies, Santa Clara, CA, USA) were used to inoculate 5 mL overnight culture of LB which was added to 100 mL 2×YT media and incubated at 37 °C and 280 rpm until a OD600 of ~2 when the 100 mL were transferred to 900 mL 2×YT. To induce β-gal omega fragment expression, IPTG was added to a final media concentration of 1 mM at OD600 of ~0.5 and cells were harvested by centrifugation at 6000 RCF for 10 min. Cells were homogenized at 20,000 psi with an Avestin Emulsiflex B15 (Avestin, Ottawa, ON, Canada). The resulting lysate was clarified by centrifugation at 12,000 RCF for 30 min and the supernatant, referred to as cell extract, was flash frozen and stored at −80 °C.
For XL1-Blue CFPS reactions requiring exogenously added T7 RNA Polymerase, T7 RNAP was expressed in E. coli BL21 DE3 STARTM cells with a pHT-T7RNAP-HIS plasmid and purified with the Akta Start Protein Purification System (GE Healthcare Bio-Sciences, Uppsala, Sweden) according to the manufacturer’s instructions. The purified formulation was added to CFPS reactions at an optimized concentration.
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Determination of cellular dry weight occurred by pelleting 2 mL samples (12,000 g for 10 min), washing cells with ddH2O and freeze drying at −80 °C for 24 h in pre-weighed microtubes. Cellular total lipid was obtained by extraction with chloroform and methanol by Folch et al. [11 (link)] (adapted). Cell pellets from 12 mL culture were washed with ddH2O twice and disrupted four times by french press (EmulsiFlex®-B15, Avestin) at 2400 bar. A triplicate of 4 mL cell lysate was transferred to glass vials with screw caps and mixed with 6 mL of Folch reagent (2:1 chloroform/methanol) each. After extraction by shaking at 900 rpm and room temperature for 1 h, 1 mL 0.9% NaCl was added to aid phase separation. Samples were vortexed, centrifuged at 1000 g and the chloroform phase was transferred to pre-weighed glass vials. After evaporation of the solvent und a constant stream of dried nitrogen, vials were weighed and lipid content was calculated per dry weight in % g/g.
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BSA-Cur-NPs and ICG-BSA-Cur-NPs were prepared using nanoparticle albumin-bound (nabTM) technology with some adjustments.39 (link) Briefly, aliquots of 50 mg BSA/1.5 mg ICG or 50 mg BSA were dissolved in 5 mL deionized water (DW). Aliquots (2 mg) of Cur was dissolved in 200 μL of a 9:1 solution of chloroform and ethanol. These two solutions were gently mixed and crudely homogenized by using a Wise Tis homogenizer HG-15D (DAIHAN Scientific Co, Seoul, South Korea) at 10,000 rpm and then by passing them through a high-pressure homogenizer (EmulsiFlex-B15 device Avestin, Ottawa, Ontario, Canada) for nine cycles at 20,000 psi. After removal of chloroform by rotary evaporator at 40°C for 15 min under reduced pressure, the resulting NPs were mildly centrifuged at 6000 rpm. The supernatant was collected and purified with Ultra centrifugal filter units (MWCO: 100 kDa, Amicon® Ultra, Millipore) to remove the unbound ICG and Cur, and was then lyophilized and stored at −20°C until required.
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To prepare nanosized LSW-ET materials, a high-pressure microfluidics technique was used as follows. LSW-ET powder was dissolved in the oil phase (72% sesame oil, 6% egg yolk lecithin) and heated to 60°C. The oil phase was mixed with the liquid phase (1.25% Tween 80, 2.81% glycerol, 95.94% water) at a ratio of 1:4 at 60°C, followed by high speed shearing at 10,000 rpm for 5 min. Furthermore, the formed coarse emulsion was then homogenized by passing it through an Avestin high-pressure homogenizer EmulsiFlex-B15 (Ottawa, Canada) for 5 cycles at 1000 bars. After cooling down to room temperature, the dispersion was filter-sterilized (a 0.22 µm cellulose acetate filter; (Zhao et al., 2015a (link)). The final concentrations were 24.089 mg/ml LSW in nano-LSW-high and 4.818 mg/ml LSW in nano-LSW-low. Nanovehicle was also prepared using the same procedure but lacking LSW-ET powder.
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Mutant METTL5 was generated using a QuikChange Site‐Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The recombinant plasmid was transformed into competent E. coli BL21 (DE3) (Bioteke, Beijing, China), and the positive monoclones were inoculated into LB‐Amp medium (Solarbio). The transformed bacteria were placed on a shaker (37°C, 100 rpm) for 3‐5 h until they reached an optical density (OD) value of 0.6‐0.8. Isopropyl β‐D‐thiogalactoside (IPTG; Solarbio) was added to induce protein expression, and the cultures were allowed to grow overnight at 16°C and 180 rpm. The next day, the bacteria were collected and lysed using an EmulsiFlex B‐15 high‐pressure homogenizer (Avestin, Ottawa, ON, Canada). Then, the lysate was centrifuged at 14000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant was mixed with Ni‐NTA resin (Qiagen Inc., Valencia, CA, USA) and incubated at 4°C for more than 40 min. After extensive washing with buffers containing 10 mmol/L, 20 mmol/L and 50 mmol/L imidazole (Solarbio), the protein was eluted in buffer containing 250 mmol/L imidazole. The protein was purified using an AKTA system (AKTA purifier, GE Healthcare, Northampton, MA, USA).
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Tau construct K19, containing three repeats, R1, R3, and R4 was purified as described by Barghorn et al. (2005) (link). In short, BL21 (Gold) were used as a host for expressing tau. Cells grown in 1 L LB broth were lysed in 10 ml of lyses buffer (50 mM Tris pH 7.4, 1 mM EDTA, 0.2 mM MgCl2, 5 mM DTT, 6 μl/ml saturated phenylmethylsulfonyl fluoride (PMSF), and 1 tablet Complete proteinase inhibitors (Roche) Cells were lysed in the high-pressure homogenizer (EmulsiFlex-B15, Avestin) and centrifuged at 30,000 ×g for 30 min. After adding NaCl to final concentration of 0.5 M, the supernatant was boiled for 20 min. Then the boiled sample was centrifuged at 20,000 ×g for 20 min. The supernatant was dialyzed against 10 mM Na phosphate pH 7.0 overnight. Next day the dialyzed protein was loaded on SP column (GE Healthcare), using 20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 1 mM MgCl2, 2 mM DTT, and 20 mM MES, pH 6.8, 1 M NaCl, 1 mM EDTA, 1 mM MgCl2, and 2 mM DTT, as elution buffer. After concentration, the protein was additionally purified on Superdex75 column equilibrated with 25 mM Na phosphate pH 7.4, 150 mM NaCl, and 1 mM DTT. Concentrations were determined by BCA Protein Assay Kit (Pierce).
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Heterologous expression of LBO in E. coli was carried out as described previously (Brewer et al., 2016). Briefly, transformed colonies were grown in LB media (0.5% yeast extract, 1% Bacto Tryptone, 1% NaCl) with carbenicillin (100 μg/ml) at 37°C in a shaking incubator (180 rpm) until the cell density reached an OD600 of 0.5–0.8. After isopropyl ‐D‐1‐thiogalactopyranoside (1 mM) was added, transformed E. coli were incubated at 20°C for 14–16 hr. To prepare enzyme fractions, E. coli cells were collected by centrifugation of 10,000 g for 1 min and suspended in 20 mM phosphate buffer (pH 7.4). The suspend cells were mechanically lysed by using a high‐pressure homogenizer (Emulsi Flex B15; AVESTIN) and then centrifuged at 15,000 g for 5 min at 4°C.
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9

Overexpression and Purification of GH13 and GH57 Glycoside Hydrolases

Codon optimized genes (glgB13 and glgB57) encoding the GH13 (PmGBE13) and GH57 (PmGBE57) GBE of P. mobilis SJ95 were synthesized by GenScript, and cloned into the NdeI-BamHI sites of the pET28a vector (Novagen), with a 6×His-tag fused at the C-terminal. Sequence details are provided in the supplemental information. glgB13 and glgB57 were overexpressed in Escherichia coli BL21(DE3) cultivated in Luria-Bertani (LB) medium (10 g/L of tryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplemented with 50 μg/mL kanamycin at 16°C for 20 h and 150 rpm. Cells were harvested by centrifugation (5,000×g, 10 min, 4°C). Cells were washed twice with 10 mM sodium phosphate buffer pH 7.0 and lysed by high-pressure homogenizer (Emulsiflex-B15; Avestin). The cell free extract was collected by centrifugation (20,000×g, 20 min, 4°C). PmGBE13 and PmGBE57 were purified in two steps. Firstly, the cell free extracts were heated at 65°C for 20 min, followed by removal of the denatured proteins by centrifugation (20,000×g, 20 min, 4°C). Subsequently, the His-tagged proteins were purified using HisPurTM Ni-NTA Resin (ThermoFisher Scientific) according to the manufacturer’s protocol. Protein concentration was quantified using the Quick Start Bradford Protein Assay kit (Bio-Rad Laboratories). Purity and molecular mass of the proteins were checked by SDS-PAGE.
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LS was expressed in vitro using a modified PANOx-SP CFPS system described previously32 (link),56 (link),57 with extract prepared using an Avestin EmulsiFlex-B15 homogenizer. After incubating the 15 µl reaction for 3 h at 30°C, geranyl pyrophospahte (GPP) was added to a final concentration of 360 µM and final volume of 25 µl. A dodecane overlay of 25 µl was added and limonene measured over time. After validation of synthase activity, the sequence was cloned into pETBCS for preparation of enriched lysates.
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