E. timonensis SN18 was purchased from Leibniz Institute DSMZ. E. timonensis SN18 culturing was performed in Hungate or Balch tubes (Chemglass Life Sciences) at 37 °C and set up in an anaerobic chamber (Coy Laboratory Products) under an atmosphere of 2% to 4% H2, 2% to 4% CO2, and N2 as the balance. Standard cultures were grown in peptone-yeast-glucose (PYG) medium, modified (medium recipe DSM 104, DSMZ Germany) that was sparged with N2 after autoclaving. Basal medium lacking electron acceptors was prepared as described previously (57 (link)), containing 1 g/L tryptone (trypticase peptone; BD Biosciences), 1 g/L yeast extract (BD Biosciences), 0.4 mM l -cysteine, 2.5 g/L NaHCO3, 1 g/L NaCl, 0.5 g/L MgCl2•6H2O, 0.2 g/L KH2PO4, 0.3 g/L NH4Cl, 0.3 g/L KCl, 0.015 g/L CaCl2•2H2O, 0.25 mL/L of 0.1% resazurin, and 1% ATCC vitamins and trace mineral solutions (ATCC). NCE medium lacking carbon sources was prepared as described previously (59 (link)), containing 4 g/L KH2PO4, 5 g/L K2HPO4, 3.5 g/L NaNH4PO4 40 mM sodium fumarate dibasic, 1 mM MgSO4•7H2O, 0.1% casamino acids (VWR Life Science), and 1% ATCC vitamin and trace mineral solutions (ATCC). All chemicals were purchased from Sigma-Aldrich unless otherwise indicated.
Casamino acids
Manufactured by Avantor
Sourced in United States
Casamino acids are a mixture of amino acids derived from the enzymatic hydrolysis of casein, a protein found in milk. They provide a readily available source of amino acids for use in microbial growth media and other applications requiring a defined amino acid profile.
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Lab products found in correlation
Yeast extract, by Thermo Fisher Scientific (2 mentions)
K2hpo4, by Avantor (1 mentions)
Yeast extract, by BD (1 mentions)
Anaerobic chamber, by Coy Laboratory Products (1 mentions)
Kh2po4, by Avantor (1 mentions)
Mgso4 7h2o, by Avantor (1 mentions)
Kh2po4, by BD (1 mentions)
Middlebrook 7h9 medium, by BD (1 mentions)
Middlebrook 7h10 agar, by BD (1 mentions)
Casamino acid, by BD (1 mentions)
Luria bertani broth, by BD (1 mentions)
Tryptone, by BD (1 mentions)
Modified tryptone soya broth, by Thermo Fisher Scientific (1 mentions)
Novobiocin, by Merck Group (1 mentions)
Yeast extract, by Hardy Diagnostics (1 mentions)
Kanamycin kan, by Merck Group (1 mentions)
Ampicillin ap, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Pvdf syringe filter, by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
11 protocols using casamino acids
1
Cultivation and Growth of E. timonensis SN18
2
Attenuated Mycobacterium tuberculosis Strain
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All experiments were performed with the attenuated Mycobacterium tuberculosis (Mtb) strain H37Rv mc26020 (ΔlysA ΔpanCD; gift of Dr. William Jacobs)65 (link). Mtb was cultured in Middlebrook 7H9 medium (BD) supplemented with 10% v/v oleic acid-albumin-dextrose-catalase (OADC) supplement (BD), 0.5% v/v glycerol, 0.2% w/v casamino acids (VWR J851), 80 mg/L L-lysine, 24 mg/L pantothenate, and 0.025% v/v Tyloxapol or on Middlebrook 7H10 agar (BD) supplemented with 10% v/v OADC, 0.5% v/v glycerol, 0.2% casamino acids, 80 mg/L L-lysine, 24 mg/L pantothenate. Mtb strains harboring APEX2-expressing plasmids were selected on medium containing 25 μg/mL kanamycin. Mtb was cultured at 37 °C and in liquid medium with shaking at 110 rpm.
3
Bacterial Strains and Phage Cultivation
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Bacterial strains, bacteriophages and plasmids used in this study are listed in Table 3 . The E. coli O157:H7 C7927 bacterial strain was used for the cultivation of phage ΦV1032 (link). Homologous recombination was carried out in a lysogenic strain E. coli O157:H7 C7927 (ΦV10) bearing the lambda Red expression plasmid pKD46. For the construction of ΦV10 reporter phage, bacterial strains were grown in Luria-Bertani (LB) broth (Difco Laboratories, MI) or LB agar plates supplemented with antibiotics as needed [ampicillin (Ap), 100 μg ml−1; kanamycin (Kan), 50 μg ml−1 (Sigma-Aldrich, MO)]. Salt-Optimized Carbon broth medium (SOC) (Clontech Laboratories, Inc, CA) was used for recovery of cells after electroporation. Modified tryptone soya broth (Oxoid Ltd., UK) containing 1% casamino acids (VWR International, PA) with novobiocin (Sigma-Aldrich, MO) of 8 mg l−1 (mTSB + n) was used for ground beef enrichment as specified by the USDA-FSIS protocol33 34 . Cell dilutions were done in phosphate buffered saline (PBS) (8 mM Na2HPO4, 6 mM NaH2PO4, 145 mM NaCl, pH7.6). Phage buffer (50 mM Tris, 100 mM MgCl2, pH7.6) was used to dilute and preserve the phage stock. LB Top agar (1% (wt/vol) tryptone (Becton Dickinson, NJ), 1% (wt/vol) NaCl (Macron, PA), 0.5% (wt/vol) yeast extract (Hardy Diagnostics, CA) and 0.6% (wt/vol) agar (Alfa Aesar, MA)) was used for the overlay plaque assay.
4
Staphylococcus aureus Culture Supernatants
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S. aureus culture supernatants (CS) were generated in (i) CCY: 3% yeast extract (Fisher BioReagents) supplemented with 2% bacto-casamino acids (Amresco), 2.3% sodium pyruvate (Fisher BioReagents), 0.63% Na2HPO4 (Fisher BioReagents) and 0.041% KH2PO4 (Sigma), (ii) BHI (Fluka) and (iii) RPMI-CAS (RPMI-1640 medium (Gibco) supplemented with 1% casamino acids (Amresco). Overnight cultures obtained from a single colony were diluted to OD600nm = 0.03 in fresh culture medium and grown to early stationary phase for 8 hours. All tested S. aureus strains reached similar bacterial densities in the respective media. CS were generated by centrifugation (5000 × g, 4 °C), followed by filter sterilization (0.1 µm pore size PVDF syringe filters; Millipore).
5
Chemotaxis Assay of Pseudomonas aeruginosa Strains
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The strains used in this study are listed in Table 1 and Supplementary Table S1 . PAO1 and the 24 MCP single mutants of PAO1 were obtained from Dr. Junichi Kato (Hiroshima University, Japan). The clinical isolates of Pa from acute infections and one environmental strain were kindly provided by Dr. Joseph D. Schwartzman and Dr. Michael Zegans (Geisel School of Medicine, Dartmouth College). Prior to being used in chemotaxis assays, all strains were incubated overnight at 37 °C in minimal salts medium (MSB) [21 (link)] supplemented with 0.5% (w/v) casamino acids (Amresco, Solon, OH, USA) and 27.5 mM glucose.
6
Mycobacterial Culture Conditions and Genetic Modification
7
Optimizing LukED Expression in S. aureus
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Staphylococcus aureus strain Newman was cultured at 37°C in yeast extract-Casamino Acids-pyruvate (YCP) medium [3% (w/v) yeast extract (Oxoid), 2% (w/v) Casamino Acids (Amresco, Washington, DC, United States), 2% (w/v) sodium pyruvate (Sangon Biotech, Shanghai, China), 0.25% (w/v) Na2HPO4, and 0.042% (w/v) KH2PO4, pH 7.0)], which is able to promote the highest expression of LukED (Alonzo and Torres, 2014 (link)).
8
Disodium Cromoglycate Synthesis Protocol
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Disodium cromoglycate (5′DSCG) (98% purity) was obtained from TCI Chemicals (Philadelphia, PA). Tryptone, yeast extract, sodium chloride, Tris base, urea, lauryldimethylamine N-oxide and poly(ethylene) glycol (PEG8000) were purchased from Fisher Scientific (Fair Lawn, NJ) and were used to make Luria Bertani media and buffers. All aqueous solutions were dissolved in deionized water with resistivity greater than 18.2 MΩ cm or deuterium oxide (D2O, 99.9%, Cambridge Isotope Laboratories, Inc., Andover, MA). Casamino acids (Amresco, Solon, OH), peptone (Bio Basic, Amherst, NY) and casitone (BD Biosciences, MD) were also used. l -amino acids (l -glutamic acid, l -alanine, l -arginine), polyvinylpyrrolidone (PVP, MW ∼ 40 000), polyvinylalcohol (PVA, MW ∼ 9000–10 000), poly-acrylamide (PAAm, MW ∼ 9000–10 000), poly(ethylene) glycol (PEG4000), and proteins (lectin A, esterase, lipase, bovine serum albumin and trypsin) were purchased from Sigma Aldrich (St. Louis, MO). Pseudomonas aeruginosa strain PA1244N3 (pPAC46) was obtained from Dr Castric and Dr Horzempa.28,29 (link)
9
Culturing E. coli and Msm Strains
10
Cultivation and Maintenance of Escherichia coli
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Escherichia coli NEB 5-alpha (New England Biolabs, Ipswich, MA, USA), E. coli S17-1 and E. coli O157:H7 Δstx (NCTC 12900) were routinely grown on Lysogeny broth (LB) or LB agar at 37°C. To prevent plasmids loss, all pGRG36 plasmid-carrying cells were grown at 32°C. For counterselection of auxotroph E. coli S17-1 after mating, MM2 medium agar (4 g L−1 L-asparagine, 2 g L 1 K2HPO4, 0.2 g L−1 MgSO4, 3 g L−1 NaCl, 10 g L−1 sorbitol, 15 g L−1 agar) was used. Where necessary, media were supplemented with 100 μg mL−1 ampicillin, 50 μg mL−1 kanamycin or 20 μg mL−1 tetracycline. For microtiter plate reader experiments, cells were grown in M9 minimal medium (20 mL L−1 20% (w/v) casamino acids (Amresco), 40 mL L−1 10% (w/v) carbon source, 2 mL L−1 1 M MgSO4, 1 mL L−1 0.1 M CaCl2, 100 mL L−1 10 × M9 salts (85.1 g L−1 Na2HPO4 × 2 H2O, 30 g L−1 KH2PO4, 5 g L−1 NaCl, 10 g L−1 NH4Cl, pH 7)) containing either glucose or lactose as sole carbon source.
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Escherichia coli NEB 5-alpha (New England Biolabs, Ipswich, MA, USA), E. coli S17-1 and E. coli O157:H7 Δstx (NCTC 12900) were routinely grown on Lysogeny broth (LB) or LB agar at 37°C. To prevent plasmids loss, all pGRG36 plasmid-carrying cells were grown at 32°C. For counterselection of auxotroph E. coli S17-1 after mating, MM2 medium agar (4 g L−1 L-asparagine, 2 g L 1 K2HPO4, 0.2 g L−1 MgSO4, 3 g L−1 NaCl, 10 g L−1 sorbitol, 15 g L−1 agar) was used. Where necessary, media were supplemented with 100 μg mL−1 ampicillin, 50 μg mL−1 kanamycin or 20 μg mL−1 tetracycline. For microtiter plate reader experiments, cells were grown in M9 minimal medium (20 mL L−1 20% (w/v) casamino acids (Amresco), 40 mL L−1 10% (w/v) carbon source, 2 mL L−1 1 M MgSO4, 1 mL L−1 0.1 M CaCl2, 100 mL L−1 10 × M9 salts (85.1 g L−1 Na2HPO4 × 2 H2O, 30 g L−1 KH2PO4, 5 g L−1 NaCl, 10 g L−1 NH4Cl, pH 7)) containing either glucose or lactose as sole carbon source.
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