Cells were grown in the bioreactor for 5 days and then centrifuged and washed twice with water to remove residual medium. Cells were resuspended in water and aliquots were transferred into 50 mL plastic screw-top vials. The vials were centrifuged to yield pellets the volumes of which (ca. 5 mL) were determined from the height of the pellet and the mass of water needed to fill the vial to that height (after the experiment). The volume of the pellet was corrected for packing efficiency. Various treatment buffers (40 mL) were added to each tube at time 0, including: water, 100 μM EDTA (prepared in 150 μM Tris buffer pH 8.0), and 100 μM EDTA plus lyticase (Sigma; 1000 U of lyticase activity per gram of wet packed cell in tube) in SP buffer (1.2 M sorbitol in 50 mM potassium phosphate pH 7.4). At 10 min intervals, 2 mL of solution were removed from each tube and transferred to microfuge tubes and centrifuged. Supernatants were saved for metal analysis and pellets were washed twice with triple-distilled water or SP buffer (lyticase samples) that lacked chelators or lyticase. The volumes of these pellets and supernatants were measured, and Fe, Mn, Cu, and Zn concentrations were determined. At each buffer condition, the relationship Vpell•[Fepell] + Vsup•[Fesup] equalled a constant number of moles at each time point within an error of ± 10%.
Lyticase
Manufactured by Merck Group
Sourced in United States, Germany, China, France
Lyticase is a laboratory enzyme used for the digestion of bacterial cell walls. It is derived from the fungus Arthrobacter luteus and is commonly used in molecular biology and microbiology experiments to facilitate the extraction of cellular contents, including DNA and RNA.
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Lab products found in correlation
Lysozyme, by Merck Group (8 mentions)
Qiaamp dna stool mini kit, by Qiagen (7 mentions)
β mercaptoethanol, by Merck Group (6 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Proteinase k, by Merck Group (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Maxwell rsc purefood gmo and authentication kit, by Promega (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Lysozyme, by Thermo Fisher Scientific (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Phenol, by Merck Group (2 mentions)
Phenol chloroform isoamyl alcohol, by Merck Group (2 mentions)
Glass bead, by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
Chitinase, by Merck Group (2 mentions)
97 protocols using lyticase
1
Cellular Metal Partitioning Assay
2
Optimizing DNA Extraction through Lyticase
3
Extraction and Preparation of Fungal Spores
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To release spores from asci, the asci were suspended in 5 mL of lyticase (β-glucanase) buffer (50 mM potassium phosphate buffer, pH 7.5, 1.4 M sorbitol), and 20 μL of lyticase (Sigma-Aldrich, St. Louis, MO, USA) stock solution (10,000 U/mL was dissolved in 500 μL of 50% glycerol) was added. After a 3 h incubation at 37 °C, the spores were washed twice with lyticase buffer. Then, the spores were resuspended in water and sonicated with an ultrasonic disruptor (Xinchen Biological Technology, Nanjing, China) to rupture the asci membrane. The sonication conditions were as follows: power—45%; duration—5 min, with cycles of 5 s on and 2 s off. Then, the spores were washed 3 times with 0.5% Tween-20 and 2 times with water. The number of spores was counted with a platelet counter.
Spores treated with high salt were prepared by incubating the spores in 0.6 M NaCl solution for 1 min, and the supernatant was removed. This process was repeated three times and then washed twice with water. To treat the spores with RNase A, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL RNase A (TaKaRa, Tokyo, Japan) were incubated at 37 °C for 1 h. Spores were washed twice with water. To prepare protease-treated spores, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL protease (Sigma-Aldrich) were incubated at 37 °C for 1 h and washed twice with water.
Spores treated with high salt were prepared by incubating the spores in 0.6 M NaCl solution for 1 min, and the supernatant was removed. This process was repeated three times and then washed twice with water. To treat the spores with RNase A, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL RNase A (TaKaRa, Tokyo, Japan) were incubated at 37 °C for 1 h. Spores were washed twice with water. To prepare protease-treated spores, 2 × 108 spores suspended in 1 mL of water supplemented with 30 U/mL protease (Sigma-Aldrich) were incubated at 37 °C for 1 h and washed twice with water.
4
Culture-independent DNA extraction from grapes and yeast
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The initial step for our culture independent approach was the extraction of DNA directly from grapevines. The pellets obtained after grape juice centrifugation were re-hydrated with 480 μL Phosphate-buffered saline (PBS), with vigorous agitation. A 20 μL aliquot of 20 mg/mL lyticase (Sigma) was added to the samples, which were subsequently incubated at 37°C for 20 min. Then, the samples were treated with 2.5 μL volume of 20 mg/mL Proteinase K (Merck) incubated at 37°C for 45 min. The Power Soil DNA Isolation Kit (Mo-Bio Laboratories, Inc.) was used for DNA extraction according to the manufacturer’s instructions.
In the case of yeast isolates, each of the colonies selected was suspended in 200 μL PBS, with vigorous agitation, followed by centrifugation at 5.000 × g for 5 min. The pellets formed were washed with TE-NaCl (Tris 10mM pH7, EDTA 1 mM, NaCl 0.15 M) and centrifuged at 5.000 × g for 5 min. Subsequently, a 20 μL volume of 20 mg/mL lyticase (Sigma) was added to the samples, which were subsequently incubated at 37°C for 20 min. Finally, the samples were treated with 2.5 μL volume of 20 mg/mL Proteinase K (Merck) incubated at 37°C for 45 min. The Power Soil DNA Isolation Kit (Mo-Bio Laboratories, Inc.) was used for DNA extraction according to the manufacturer’s instructions. All the DNA obtained was froze at -20°C until processed.
In the case of yeast isolates, each of the colonies selected was suspended in 200 μL PBS, with vigorous agitation, followed by centrifugation at 5.000 × g for 5 min. The pellets formed were washed with TE-NaCl (Tris 10mM pH7, EDTA 1 mM, NaCl 0.15 M) and centrifuged at 5.000 × g for 5 min. Subsequently, a 20 μL volume of 20 mg/mL lyticase (Sigma) was added to the samples, which were subsequently incubated at 37°C for 20 min. Finally, the samples were treated with 2.5 μL volume of 20 mg/mL Proteinase K (Merck) incubated at 37°C for 45 min. The Power Soil DNA Isolation Kit (Mo-Bio Laboratories, Inc.) was used for DNA extraction according to the manufacturer’s instructions. All the DNA obtained was froze at -20°C until processed.
5
Yeast Cell Lysis and Protein Extraction
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The cells were collected by centrifugation (10,000×g, 1 min) and then incubated in 1 ml lyticase buffer (1.0 M sorbitol, 2 mM MgCl2, 0.14 % β-mercaptoethanol, 50 mM Tris–HCl, pH 7.5) with lyticase (Sigma-Aldrich, Shanghai, China) at a concentration of 50 U/OD600. After incubation (30 °C, 60 min) most of the cells were converted to spheroplasts. The spheroplasts were collected and washed twice with ice-cold lysis buffer (0.2 M sorbitol, 1 mM EDTA, 50 mM Tris–HCl, pH 7.5). Spheroplasts were then suspended in 500 μl lysis buffer containing protease inhibitors (1 mM PMSF) and an appropriate amount of glass beads. The mixture was incubated on ice for 30 s and vortexed 30 s for about five to seven cycles (URBomix VORTEX-GENIE, Scientific Industries, USA). Cells were lysed and centrifuged (1000×g, 4 °C) for 10 min to remove unlysed cells and cell wall debris. The suspensions were carefully collected as whole intracellular protein. Protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Jiangsu, China). About 10 μg of protein was separated via SDS-PAGE for western blotting with anti-CPY antibody (Thermo Scientific, Waltham, USA) as described below.
6
Spheroplast Formation and Cell Lysis
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In total, 25–50 ml of cell culture with density at 1-2×10
7 cells/ml, was pelleted and resuspended in 12.5 ml of solution 1 (0.1 M PIPES/KOH pH 9.4, 10 mM DTT) and incubated at 30°C for 10 minutes. The cells were pelleted and resuspended in 5 ml of solution 2 (2xSC media with 4% glucose, 0.6 M Sorbitol, 25 mM Tris, pH 7.5). Then, 25 μl of lyticase (1.2 M sorbitol, 0.1 M, Sodium Phosphate pH 7.4, 50% (v/v) glycerol, 50 μl/ml β-mercaptoethanol, 40 KU/ml lyticase (L5263, Sigma) was added and the solution was incubated at 30°C for 5 minutes with mild agitation. Spheroplast formation was checked microscopically and the cells were then pelleted at 200 g (~1,000 rpm on bench centrifuge) for 3 minutes. The pellet was resuspended in solution 3 (2 x SC media with 4% glucose, 0.7 M Sorbitol, 25 mM Tris, pH 7.5) and incubated at 30°C for 10 minutes with mild agitation. The spheroplasts were pelleted again at 200 g lysed in 400 μl of RIPA buffer (0.5% SDS, 50 mM Tris pH 7.5, 150 Mm NaCl, 0.5% Sodium deoxycholate, 1% Triton X-100). The rest of the protocol is the same as bead enrichment using whole cell lysates.
7 cells/ml, was pelleted and resuspended in 12.5 ml of solution 1 (0.1 M PIPES/KOH pH 9.4, 10 mM DTT) and incubated at 30°C for 10 minutes. The cells were pelleted and resuspended in 5 ml of solution 2 (2xSC media with 4% glucose, 0.6 M Sorbitol, 25 mM Tris, pH 7.5). Then, 25 μl of lyticase (1.2 M sorbitol, 0.1 M, Sodium Phosphate pH 7.4, 50% (v/v) glycerol, 50 μl/ml β-mercaptoethanol, 40 KU/ml lyticase (L5263, Sigma) was added and the solution was incubated at 30°C for 5 minutes with mild agitation. Spheroplast formation was checked microscopically and the cells were then pelleted at 200 g (~1,000 rpm on bench centrifuge) for 3 minutes. The pellet was resuspended in solution 3 (2 x SC media with 4% glucose, 0.7 M Sorbitol, 25 mM Tris, pH 7.5) and incubated at 30°C for 10 minutes with mild agitation. The spheroplasts were pelleted again at 200 g lysed in 400 μl of RIPA buffer (0.5% SDS, 50 mM Tris pH 7.5, 150 Mm NaCl, 0.5% Sodium deoxycholate, 1% Triton X-100). The rest of the protocol is the same as bead enrichment using whole cell lysates.
7
Yeast Cell Lysis for DNA Extraction
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Lyticase (Sigma-Aldrich, France) is a synergistic enzyme complex of endoglucanase and protease that catalyzes yeast cell lysis by β-1,3-glucanase (linear glucose polymers at beta.-1,3-linkages) and a highly specific alkaline protease activity, producing protoplasts or spheroplasts [33] (link).
Bacterial or fungal suspensions (samples + IC) were mixed with tris-EDTA buffer and 10 U recombinant Lyticase/100 µl of suspension and incubated at 37°C for 60 min. The suspensions were vortexed thoroughly, heated for 10 min at 94°C, cooled and 100 µl were used for DNA extraction [30] , [34] (link).
Bacterial or fungal suspensions (samples + IC) were mixed with tris-EDTA buffer and 10 U recombinant Lyticase/100 µl of suspension and incubated at 37°C for 60 min. The suspensions were vortexed thoroughly, heated for 10 min at 94°C, cooled and 100 µl were used for DNA extraction [30] , [34] (link).
8
Yeast Cell Adhesion and Fixation Protocol
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Yeast culture in sorbitol medium was placed on ConA-coated coverslips and incubated for 30 min at room temperate. After washing with PBS, the coverslip with the sample was fixed for 30 min in 4% formaldehyde (157-8, Electron Microscopy Sciences, Hatfield, PA) at 4 • C and unbound cells were removed by washing more than three times with phosphate-buffered saline (PBS, 311-010-CL, Wisent, Quebec, Canada). Add 50 µL of digestion solution containing Lyticase (Lyticase, L2524, Sigma-Aldrich, St. Louis, MO, USA), 1 M sorbitol, and 50 mM Trisbuffer (Tris, A610195, Sangon Biotech, Shanghai, China) to each sample, and the mixture was incubated for 30 min at 30 • C with gentle shaking. Next, the samples were immersed in cold methanol for 5 min to stop the reaction and then incubated in acetone for 30 s. At each step, the sample was gently washed more than three times in PBS. All samples were inspected with microscopy to confirm the number of attached yeast cells before labeling.
9
Crude Mitochondrial Isolation from Yeast
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Crude mitochondrial isolation was performed as described previously (Van Vranken et al., 2018 (link)). Cell pellets were resuspended in TD buffer (100 mM Tris–SO4, pH 9.4 and 100 mM DTT) and incubated for 15 min at 30°C. Cells were then washed once in SP buffer (1.2 M sorbitol and 20 mM potassium phosphate, pH 7.4) and incubated in SP buffer with 0.3 mg/ml lyticase (Sigma, L4025) for 1 hr at 30°C to digest the cell wall. Spheroplasts were washed once and homogenized in ice-cold SEH buffer (0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.4, 1 mM PMSF, yeast protease inhibitor cocktail [Sigma, P8215]) by applying 20 strokes in a dounce homogenizer. Crude mitochondria were isolated by differential centrifugation at 3000 × g first to remove larger debris and 10,000 × g to pellet down mitochondria. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, 23225).
10
Optimized Fecal DNA Extraction for Fungal Metagenomics
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Fecal DNA was extracted by using Maxwell® RSC PureFood GMO and Authentication Kit (Promega) with some modifications to increase the yield of fungi DNA. Approximately 100 mg from each stool sample was prewashed with 1 ml ddH2O and pelleted by centrifugation at 13,000 × g for 1 min. The pellet was resuspended in 800 μl TE buffer (pH 7.5), supplemented with 1.6 μl 2-mercaptoethanol and 500 U lyticase (Sigma) digesting cell walls of fungi, and incubated at 37 °C for 60 min, which increase the lysis efficacy of fungal cell. The sample was then centrifuged at 13,000 × g for 2 min and the supernatant was discarded. After this pretreatment, DNA was subsequently extracted from the pellet using a Maxwell® RSC PureFood GMO and Authentication Kit (Promega) following manufacturer’s instructions. Briefly, 1 ml of CTAB buffer was added to the pellet and vortexed for 30 s, then the solution heated at 95 °C for 5 min. After that, samples were vortexed thoroughly with beads (Biospec, 0.5 mm for fungi and 0.1 mm for bacteria,1:1) at maximum speed for 15 min. Following this, 40 µl proteinase K and 20 µl RNase A were added and the mixture Incubated at 70 °C for 10 min. The supernatant was then obtained by centrifuging at 13,000 × g for 5 min and placed in a Maxwell® RSC instrument for DNA extraction. The extracted fecal DNA was used to preform metagenomics sequencing.
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