For the fermentation experiments, 5 mL of each hydrolysate was pipetted into sterile serum bottles and degassed overnight in an anaerobic chamber. A culture of either Z. mobilis ZM2032 [53 (link)] or S. cerevisiae GLBRCY945 [54 (link)] was grown overnight and diluted into anaerobic media the day of the experiment. Once the cultures reached logarithmic growth, they were centrifuged, and cell pellets were resuspended with synthetic medium [44 (link)], and inoculated into 60 mL Wheaton serum bottles. The serum bottles were capped with airtight Chemglass Life Sciences Blue Butyl, 20 mm rubber caps and placed on a 120 rpm shaker in a 30 °C environmental growth chamber. The cultures were attached to respirometer cartridges using BD PrecisionGlide 23GX1 (0.6 mm × 25 mm) sterile needles inserted into the serum bottle caps. The respirometer (AER-800; Challenge Technology; Springdale, AR, USA) measured the volume of gas produced by the growing culture. Each experiment was run for 48 h, unless stated otherwise. Supernatants from post-fermentation cultures were analyzed by high-performance liquid chromatography (HPLC) and refractive index detection (RID) for sugar and ethanol concentrations [52 (link)]. Final cell density (OD600) measurements were made with a Beckman DU720 spectrophotometer.
Du720 spectrophotometer
Manufactured by Beckman Coulter
Sourced in United States
The DU720 spectrophotometer is a laboratory instrument designed for the measurement of the absorbance or transmittance of light through a sample. It is capable of analyzing the light absorption or transmission properties of various substances, providing quantitative data about the composition and concentration of the sample.
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9 protocols using du720 spectrophotometer
1
Fermentation of Lignocellulosic Hydrolysates
2
Legionella pneumophila Strains and Growth
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L. pneumophila clinical isolate strain 130b (American Type Culture Collection {ATCC} BAA-74) was used as the wildtype strain for nearly all experiments [22 (link)]. Strain NU360, a lpxP mutant of strain 130b that is defective for modification to lipid A, was previously described [104 (link)]. Other wildtype strains of L. pneumophila tested for intracellular growth and/or cytokine elicitation were clinical isolates Philadelphia-1 (ATCC 33152), Paris (ATCC 700174), Chicago-8 (ATCC 33823), 82A3105 (ATCC 43736), and 1169-MN-H (ATCC 43703) [138 (link),139 (link)]. Other species of Legionella that were tested included Legionella birminghamensis strain 1407-AL-H (ATCC 700709), Legionella longbeachae strain Long Beach 4 (ATCC 33462), and Legionella micdadei Stanford-R [104 (link),139 (link),140 (link)]. Prior to infections, all L. pneumophila strains and other Legionella species were cultured for 3 days at 37°C on buffered charcoal yeast extract (BCYE) agar [141 (link)]. In order to obtain LPS samples, L. pneumophila strain 130b and its mutant were grown with shaking at 37°C in buffered yeast extract (BYE) broth, with the stage of bacterial growth determined by measuring the optical density (OD) of the cultures at 660 nm using a DU720 spectrophotometer (Beckman) [142 (link),143 (link)]. Unless noted otherwise, chemicals were purchased from Sigma-Aldrich.
3
Legionella pneumophila Extracellular Growth
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L. pneumophila colony morphology was assessed after 7 days of incubation on BCYE agar. In order to further monitor the extracellular growth of L. pneumophila strains, legionellae grown on BCYE agar were inoculated into buffered yeast extract (BYE) broth and incubated at 37°C with shaking (Tyson et al., 2013 (link)). The optical density (OD) of each culture was then determined at 660 nm using a DU720 spectrophotometer (Beckman Coulter). Cell-free supernatants collected from late-log BYE cultures were assayed for protease activity as measured by azocasein hydrolysis, for phosphatase activity as measured by the release of p-nitrophenol from p-nitrophenol phosphate, and for lipase activity as measured by the release of p-nitrophenol from p-nitrophenol caprylate (Aragon et al., 2000 (link), 2001 (link), 2002 (link)).
4
Rab and HRas GTPase Hydrolysis Assay
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GTP-hydrolysis activities of recombinant Rab and HRas proteins were assayed by quantitating released free phosphate molecules, using the Malachite Green-based reagent Biomol Green (Enzo Life Sciences). Purified Rab and HRas proteins (4 µM or 6 µM final for each) were incubated at 30°C for 2 hours in HN150 containing MgCl2 (6 mM), DTT (1 mM), and GTP (1 mM), GDP (1 mM), or GTPγS (1 mM) where indicated. The reaction mixtures (100 µl each) were then supplemented with 900 µl of the Biomol Green reagent for each, incubated at 30°C for 20 min or 30 min, and analyzed by measuring the absorbance at 650 nm with a DU720 spectrophotometer (Beckman Coulter). The heat-treated Rab and HRas GTPases that had been denatured by treatment at 100°C for 15 min were also tested with the same protocol. Data obtained in this assay were corrected by subtracting the absorbance value of the control reaction assayed in the absence of Rab and HRas proteins. Means and standard deviations of the corrected values (ΔA650) were determined from three independent experiments.
5
Turbidity Analysis of Liposome-Rab Interactions
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Turbidity of liposome suspensions was analyzed as described (Ohki et al., 1982 (link)). Liposomes (Rh-labeled liposomes, 1.3 mM or 1.0 mM lipids) were mixed with Rabs (0.5–2 µM) in HN150 containing 5 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, and 2.5% glycerol, followed by measuring the absorbance at 400 nm at room temperature in a DU720 spectrophotometer (Beckman Coulter).
6
Solubility Testing of Samples
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The solubility test was performed under different media, that is, water and 0.1 N HCl. An excess amount (2 g) of samples was added, to a test tube containing 10 mL of medium to saturate. The reaction tube was shaken in an orbital shaker (120 Hz, 37 ± 0.5 °C) for 24 h until an equilibrium condition was reached. The solution was filtered using a 0.22-μm nylon filter, and the solution was then analyzed using a Beckman Coulter DU720 spectrophotometer (Brea, CA, USA) at 290 nm wavelength using a validated analysis method.
7
Polystyrene Plate Biofilm Assay Protocol
8
Quantifying Arf GTPase Hydrolysis
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GTP hydrolysis activities of purified His12-Arf proteins were assayed by quantitating free phosphate molecules released in the hydrolytic reactions, using the Malachite Green-based reagent Biomol Green (Enzo Life Sciences) as described for the activity assays with Rab-family small GTPases (Inoshita and Mima, 2017; Segawa et al., 2019) . Purified His12-Arf1 and His12-Arf6 proteins (2 M final) were mixed with GTP or GTPS (1 mM final) in RB150 containing 5 mM MgCl 2 and 1 mM DTT (100 l each), incubated (30°C, 1 h), and then supplemented with the Malachite Green reagent (100 l each). After incubation with the reagent (30°C, 30 min), the absorbance at 620 nm (A620) of the reactions (200 l each) was measured with a DU720 spectrophotometer (Beckman Coulter). The raw A620 data were corrected by subtracting the A620 values of the control reactions without His12-Arf proteins. Concentrations of phosphate molecules released in the reactions were calculated using the corrected A620 data and the A620 values of phosphate standards (Enzo Life Sciences). Means and standard deviations of the specific GTPase activities (M phosphate/min/M protein) were determined from three independent experiments.
9
MFGM Precipitation Optimization
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The β stream powder was dispersed in deionized water (5% wt/wt) and allowed to hydrate for 4 h under constant agitation (450 rpm). The pH of the dispersion was 4.7, and 2 M sodium hydroxide solution was used to adjust the pH to a specific value (pH monitored using Thermo Fisher Scientific Orion Star A111 pH meter). After hydration, powdered zinc acetate or calcium acetate was added at the designed molar concentration (0, 25, 50, 75, 100, 150, and 200 mM) . An exception was made for studying the effect of pH on the efficiency of lipid recovery, wherein the pH was adjusted after zinc acetate or calcium acetate addition, because these salts can lower the pH of the β stream dispersion (Figure 1). The samples were then placed in a hot water bath (Thermo Fisher Scientific, model 3582) at 30 or 60°C for 30 min. After this heat treatment, the samples were centrifuged (IEC Centra CL-2, Thermo Fisher Scientific) at 1,800 × g for 5 min at ambient temperature. The samples were then allowed to cool overnight in the refrigerator (2 to 4°C), and the supernatant was then decanted and the lower pellet collected for total lipid extraction and PL quantification. The transmittance of the supernatant was measured at 500 nm using a Beckman Coulter (Brea, CA) DU 720 spectrophotometer as an indicator of effective MFGM precipitation.
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