Groups followed their own diet (HFD, HFD-LS-O, and HFD-LS-E) during the entire protocol. Pair feeding was performed throughout the protocol to ensure that groups consumed the same food quantity, as spontaneous physical activity can increase food intake [75 (link)]. Mice had access to food and drinking water ad libitum. After 12 weeks of protocol, dextran sulfate sodium (1%) (MP biomedicals, Irvin, CA, USA) was added to the drinking water. After three days of DSS, all mice were also orally challenged once with the AIEC LF82 strain (109 bacteria), isolated from a patient with CD. The AIEC LF82 strain was grown in LB broth (Condalab, Madrid, Spain), without agitation, at 37 °C for 24 h. Seven days after the AIEC LF82 strain exposition, mice were euthanized by cervical dislocation (Figure 6 ).
Lb broth
Manufactured by Condalab
Sourced in Spain
LB broth is a widely used nutrient-rich growth medium for the cultivation of various microorganisms, particularly bacteria. It provides essential nutrients and growth factors required for the optimal growth and proliferation of bacterial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Labopass tissue genomic dna mini kit, by Cosmogenetech (2 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Abi 3730xl capillary dna sequencer, by Thermo Fisher Scientific (1 mentions)
Thiostrepton, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
N n diisopropylethylamine diea, by Merck Group (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
Lb agar plates, by Condalab (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Trehalose dihydrate, by Merck Group (1 mentions)
Agarose, by Condalab (1 mentions)
Peptone, by Condalab (1 mentions)
Agarose, by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
European bacteriological agar, by Condalab (1 mentions)
Absolute ethanol, by Scharlab (1 mentions)
Sodium azide, by Merck Group (1 mentions)
9 protocols using lb broth
1
High-Fat Diet-Induced Colitis Model with AIEC Challenge
2
Identification of P. agglomerans KM1 by 16S rRNA sequencing
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A single colony of P. agglomerans KM1 was grown in LB broth (Conda, Spain) overnight at 37°C. The genomic DNA was isolated using the LaboPass™ Tissue Genomic DNA mini kit (Cosmo Genetech, Seoul, South Korea) according to the manufacturer’s protocol. The identity of the isolate was verified through 16S rRNA gene sequencing. The 16S rRNA gene was amplified from the extracted genomic DNA using 16S universal primers 27F (5’-AGA GTTTGATCMTGGCTCAG-3’ ) and 1492R (5’-GGTTACCTTGTTACGACTTC-3’ ) and sequenced using an automated ABI3730XL capillary DNA sequencer (Applied Biosystems, USA) for taxonomic identification at Cosmo Genetech (Seoul, South Korea). The 16S rRNA sequences were confirmed through BLASTn search against the NCBI microbial 16S database.
3
Bacterial Strains and Culture Conditions
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Bacterial strains, plasmids and oligonucleotides used in this work are listed in Tables 1 and 2 , respectively. Escherichia coli strains were routinely grown in LB broth (Condalab) at 37°C. R. erythropolis strains were incubated in LB broth, or Basal Salt Medium (BSM) (del Olmo et al., 2005 ) at 30°C. Media, when required, were supplemented with appropriate antibiotics at the following concentrations: ampicillin (Amp, 100 µg ml−1), chloramphenicol (Cm, 34 µg ml−1) and kanamycin (Km, 30 µg ml−1). Bacteriological agar was used as gelling agent (VWR). A stock solution of 20 mg ml−1 thiostrepton (Sigma‐Aldrich) in glacial acetic acid was prepared and added to cultures at 1 µg ml−1 to induce gene expression under the tipA promoter (PtipA).
4
Peptide Synthesis and Bacterial Strains
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Rink amide 4-methylbenzhydrylamine (MBHA) resin and N-(9-fluorenyl)methoxycarbonyl (Fmoc)-amino acids were purchased from
Calbiochem-Novabiochem. Other reagents used for peptide synthesis
included trifluoroacetic acid (TFA, Sigma-Aldrich), N,N-diisopropylethylamine (DIEA, Sigma-Aldrich),
dichloromethane (DCM, peptide synthesis grade, Bio-Lab), dimethylformamide
(DMF, peptide synthesis grade, Bio-Lab), and hydroxybenzotriazole
(HOBT) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) (peptide synthesis grade, Bio-Lab). Three S. enterica serovar Typhimurium strains ATCC 14028
(WT), the phoP-knockout derivative,44 (link) a
gift from Prof. Shoshi Altuvia’s lab (the Hebrew University,
Jerusalem), and a PmrAB knockout were used in the present study.
Kanamycin, tetracycline, and chloramphenicol were purchased from Sigma
(catalog no. P1004, K-1377, T-3383, C0378, respectively). The media
used were lysogeny broth (LB, containing 20 g/L LB broth, Conda),
SOC (26.6 g/L SOB, Conda, supplemented with 0.4% glucose, Merck),
and modified N-minimal media (MNMM, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 0.01 mM Tris–HCl pH 7.4, 1 mM MgCl2, 0.4% glucose, 38 mM glycerol, and 0.1% casamino acids).45 (link)
Calbiochem-Novabiochem. Other reagents used for peptide synthesis
included trifluoroacetic acid (TFA, Sigma-Aldrich), N,N-diisopropylethylamine (DIEA, Sigma-Aldrich),
dichloromethane (DCM, peptide synthesis grade, Bio-Lab), dimethylformamide
(DMF, peptide synthesis grade, Bio-Lab), and hydroxybenzotriazole
(HOBT) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) (peptide synthesis grade, Bio-Lab). Three S. enterica serovar Typhimurium strains ATCC 14028
(WT), the phoP-knockout derivative,44 (link) a
gift from Prof. Shoshi Altuvia’s lab (the Hebrew University,
Jerusalem), and a PmrAB knockout were used in the present study.
Kanamycin, tetracycline, and chloramphenicol were purchased from Sigma
(catalog no. P1004, K-1377, T-3383, C0378, respectively). The media
used were lysogeny broth (LB, containing 20 g/L LB broth, Conda),
SOC (26.6 g/L SOB, Conda, supplemented with 0.4% glucose, Merck),
and modified N-minimal media (MNMM, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 0.01 mM Tris–HCl pH 7.4, 1 mM MgCl2, 0.4% glucose, 38 mM glycerol, and 0.1% casamino acids).45 (link)
5
Genetic Modification of E. coli Strains
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All the E. coli strains used and constructed in the present study originate from the XTL632 strain ‘MG1655 galM<tetA-sacB>gpmA’ from the Court laboratory (20 (link)) and are listed in Supplementary Table S1 . The plasmid and oligonucleotide sequences are listed in Supplementary Tables S2 and S3 . Escherichia coli strains were grown on LB agar plates (Condalab) and in LB broth (Condalab) (with 30 μg/ml chloramphenicol when needed).
6
Genomic DNA Extraction and Whole Genome Sequencing
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For genomic DNA extraction, the strains were grown in LB broth (Condalab, Madrid, Spain) under agitation at 37 °C for 24 h. Subsequently, DNA was obtained using the Promega Wizard Genomics extraction kit (Promega Corp., Madison, WI, USA) and QIAmp® DNA Mini Kit (QIAGEN, Hilden, Germany). Whole genome sequencing was performed at the National Laboratory of Animal Digestive Nutrigenomics and Microbiomics (LANMDA-IPN) and DNA quantification was performed using the Qubit dsDNA HS Assay kit on the Qubit 3.0 fluorometer (Thermo Scientific, Waltham, MA, USA). Libraries were constructed using the Nextera Flex library kit. Libraries were sequenced using the MiniSeq™ sequencing system (150 bp paired-end reads).
7
Biochemical Composition Analysis of Bevacizumab Formulations
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Human serum albumin, sodium deoxycholate (DS), docusate sodium salt (DOCU), poly(ethylene glycol) 35,000 Da (PEG35), trehalose dihydrate, sodium azide, and agarose were purchased from Sigma-Aldrich (Steinheim, Germany). Bevacizumab (Avastin®) was purchased from Roche (Madrid, Spain). Ethanol absolute was obtained from Scharlab (Sentmenat, Spain). Lumogen® F-Red 305 was supplied by BASF (Ludwigshafen am Rhein, Germany). European bacteriological agar, peptone, LB broth, and agarose were provided by Condalab (Torrejón de Ardoz, Spain). O.C.T.™ Compound Tissue-Tek was obtained from Sakura Finetek Europe (Alphen aan Der Rijn, The Netherlands). Isoflurane was purchased from Braun (Barcelona, Spain). Shikari® Q-BEVA Enzyme Immunoassay used for the detection of bevacizumab was purchased from Matriks Biotek (Gölbaşı, Turkey).
8
Identification of P. agglomerans KM1 by 16S rRNA
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A single colony of P. agglomerans KM1 was grown in LB broth (Conda, Spain) overnight at 37 o C. The genomic DNA was isolated using the LaboPass™ Tissue Genomic DNA mini kit (Cosmo Genetech, Seoul, South Korea) according to the manufacturer's protocol. The identity of the isolate was verified through 16S rRNA gene sequencing. The 16S rRNA gene was amplified from the extracted genomic DNA using 16S universal primers 27F (5'-AGA GTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTTC-3') and sequenced using an automated ABI3730XL capillary DNA sequencer (Applied Biosystems, USA) for taxonomic identification at Cosmo Genetech (Seoul, South Korea). The 16S rRNA sequences were confirmed through BLASTn search against the NCBI microbial 16S database.
9
Isolation and Characterization of Vibrio alginolyticus
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A local V. alginolyticus isolate strain VA2 was used in this study. It was isolated from diseased tiger grouper, Epinephelus fuscoguttatus isolated from local farm in Malaysia. The isolate was grown at 30 °C on tryptic soy agar (TSA) agar (Merck, Germany) containing 1.5% sodium chloride (NaCl) and maintained in tryptic soy broth (TSB) (Merck, Germany) with 1.5% NaCl containing 20% glycerol at -80 °C until further use. The strain was identified as V. alginolyticus based on phenotypic characterizaton by colony and cell morphology, Gram stain and characterized by 16S ribosomal RNA (rRNA) gene sequencing.
Escherichia coli TOP10 (Invitrogen, USA) and E. coli BL21(DE3) (Novagen, USA) were used as cloning and expression hosts, respectively. Both strains were grown in Luria Bertani (LB) agar and LB broth (Conda, Spain) at 37 °C, with added ampicillin (50 μg mL -1 ) (Amresco, USA) whenever necessary. Overnight cultures were maintained in LB broth containing 20% glycerol at -80 °C until further use. Plasmid pET-32 Ek/LIC Vector (Novagen, USA) with His-tag was used to conserve the cloned genes for sequencing and to construct recombinant expression plasmid.
Escherichia coli TOP10 (Invitrogen, USA) and E. coli BL21(DE3) (Novagen, USA) were used as cloning and expression hosts, respectively. Both strains were grown in Luria Bertani (LB) agar and LB broth (Conda, Spain) at 37 °C, with added ampicillin (50 μg mL -1 ) (Amresco, USA) whenever necessary. Overnight cultures were maintained in LB broth containing 20% glycerol at -80 °C until further use. Plasmid pET-32 Ek/LIC Vector (Novagen, USA) with His-tag was used to conserve the cloned genes for sequencing and to construct recombinant expression plasmid.
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