Callistemon viminalis leaf was brought from Agricultural Park, Hat Yai, Songkhla-90110, Thailand. Zinc nitrate hexahydrate and Ruthenium(iii ) chloride hydrate were purchased from Sigma Aldrich, Singapore. Silver nitrate and sodium hydroxide were purchased from Loba Chem Pvt. Ltd., Thailand. Nutrient broth media, Nutrient Agar Media, Muller Hilton Agar Media, Eosin Methylene Blue (EMB) Media, and Luria Bertani (LB) Media were brought from HiMedia Laboratories Pvt. Ltd., Mumbai, India. MacConkey Agar was purchased from Becton, Dickinson, and Company, Sparks, MD 21152, USA.
Luria bertani media
Manufactured by HiMedia
Sourced in India
Luria Bertani (LB) media is a commonly used nutrient-rich growth medium for culturing bacteria. It provides essential nutrients, such as peptides, yeast extract, and salts, to support the growth and proliferation of a wide range of bacterial species.
Automatically generated - may contain errors
Lab products found in correlation
Tween 80, by Merck Group (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Apramycin, by Merck Group (2 mentions)
Zinc nitrate hexahydrate, by Merck Group (1 mentions)
Macconkey agar, by BD (1 mentions)
Nutrient broth media, by HiMedia (1 mentions)
Ruthenium 3 chloride hydrate, by Merck Group (1 mentions)
Nutrient agar media, by HiMedia (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Bio Basic (1 mentions)
Pet22b expression vector, by Merck Group (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Lb agar, by HiMedia (1 mentions)
Middlebrook 7h9 media, by BD (1 mentions)
Middlebrook adc, by BD (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Kanamycin, by AppliChem (1 mentions)
Taq dna polymerase master mix red, by Ampliqon (1 mentions)
Phusion high fidelity dna polymerase mix, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
8 protocols using luria bertani media
1
Biosynthesis of Metal Nanoparticles
2
Expression of Human DMP1 Protein
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The human DMP1 gene with a 6xHis tag at the C-terminus was synthesized and reported (Fig. 1 ). The DMP1 gene was inserted into the pET22b expression vector (Merck, USA) by using EcoRI and SacI (New England Biolabs, UK) restriction enzymes. The pET22b-DMP1 plasmid was transformed into E. coli strain Rosetta (DE3) (Novagen, Germany). The bacterial cells were cultured in Luria Bertani (LB) media (HiMedia Laboratories, India) with 100 μg/ml ampicillin (ITW Reagents, Germany) at 37 °C with shaking at 200 rpm. After the OD600 of the culture reached 0.6, protein expression was induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) (Bio Basic, Canada) to final concentrations of 0.2, 0.5, and 1 mM. After IPTG induction, the incubation continued at either 28 °C or 37 °C on a rotary shaker at 200 rpm. The cells were harvested by centrifugation every 2, 4 and 6 h and suspended in 100 μl of 1X SDS loading dye buffer (125 mM Tris HCl, pH 6.8, 12% SDS, 10% glycerol, 22% β-mercaptoethanol, and 0.001% bromophenol blue).Fig. 1 ![]()
Nucleotide and amino acid sequences of the human DMP1 gene.
3
Intracellular Pathogen Infection Assay
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M. tuberculosis strain Erdman was grown in 7H9 Middlebrook media (BD Biosciences, San Jose, CA, USA) supplemented with Middlebrook ADC (BD, USA) at 37°C. Escherichia coli and S. enterica serovar Typhimurium were grown in Luria–Bertani (LB) media (HiMedia Laboratories, Mumbai, India) at 37°C. Single-cell suspension of mid-log phase bacteria was adjusted to the required cell density and then used to infect the differentiated THP1 macrophages at a multiplicity of infection (MOI) of 5. After 6 h p.i. for Mtb, cells were washed with phosphate-buffered saline (PBS) to remove extracellular bacteria, and at various times post-infection, bacterial numbers were enumerated by lysis of cells and plating for colony-forming unit (CFU) in 7H10 agar plates. For S. typhimurium/E. coli, post-addition of bacteria at a MOI of 10, the plates were centrifuged for 5 min and then left for 20 min at 37°C. The media was removed and treated with 100 μg/ml of gentamicin for 2 h in RPMI at 37°C. The cells were washed with PBS three times, and infection was continued in media containing 12 μg/ml of gentamicin, and at indicated time points, cells were lysed with PBS–Triton X-100 (1%) for CFU plating on LB agar (HiMedia Laboratories, Mumbai, India) plates. The colonies were counted and represented as CFU/well.
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M. tuberculosis strain Erdman was grown in 7H9 Middlebrook media (BD Biosciences, San Jose, CA, USA) supplemented with Middlebrook ADC (BD, USA) at 37°C. Escherichia coli and S. enterica serovar Typhimurium were grown in Luria–Bertani (LB) media (HiMedia Laboratories, Mumbai, India) at 37°C. Single-cell suspension of mid-log phase bacteria was adjusted to the required cell density and then used to infect the differentiated THP1 macrophages at a multiplicity of infection (MOI) of 5. After 6 h p.i. for Mtb, cells were washed with phosphate-buffered saline (PBS) to remove extracellular bacteria, and at various times post-infection, bacterial numbers were enumerated by lysis of cells and plating for colony-forming unit (CFU) in 7H10 agar plates. For S. typhimurium/E. coli, post-addition of bacteria at a MOI of 10, the plates were centrifuged for 5 min and then left for 20 min at 37°C. The media was removed and treated with 100 μg/ml of gentamicin for 2 h in RPMI at 37°C. The cells were washed with PBS three times, and infection was continued in media containing 12 μg/ml of gentamicin, and at indicated time points, cells were lysed with PBS–Triton X-100 (1%) for CFU plating on LB agar (HiMedia Laboratories, Mumbai, India) plates. The colonies were counted and represented as CFU/well.
4
Production of Recombinant Human DMP1 Protein
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The plant-optimized DNA sequence encoding human DMP1 was based on a previous study [22 (link)]. The gene encoding hDMP1 with 8xHis was inserted into the geminiviral expression vector pBYR2e [31 (link)] by using the XbaI and SacI restriction enzyme (New England BioLabs, Ipswich, MA, USA). The pBYR2e-DMP1 vector (Figure 1 ) was transformed into E. coli DH10B by using the heat shock method. The selected colonies were analyzed by colony PCR and cultured in Luria Bertani (LB) media (HiMedia Laboratories, Mumbai, India) with 100 mg/mL ampicillin (ITW Reagents, Darmstadt, Germany) at 37 °C overnight.
5
Plant-Derived Anti-PD-L1 Antibody Production
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The amino acid sequences encoding Atezolizumab (Drugbank accession number: DB11595) were codon optimized in silico using GeneArt gene synthesis software (Thermo Scientific, MA, USA) for the expression of anti-PD-L1 antibody in plant system. Variable regions of Atezolizumab light chain (LC) and heavy chain (HC) that were added individually to human IgG1 kappa chain (Genbank accession number: AAA58989.1) and gamma chain (Genbank accession number: AAA02914.1) were flanked with signal peptide on the N-terminus and a SEKDEL (Ser-Glu-Lys-Asp-Glu-Leu) sequence on C-terminus. Anti-PD-L1 light chain (anti-PD-L1-LC) and anti-PD-L1 heavy chain (anti-PD-L1-HC) that are synthesized were used for cloning into the geminiviral vector pBYR2eK2Md (pBYR2e) by double digestion with XbaI and SacI restriction enzymes (BioLabs, MA, USA) separately (Fig 1 ). The constructed plant expression vectors were transformed into Escherichia coli DH10B by heat shock method. Selected colonies were confirmed by colony polymerase chain reaction (PCR) with the primers mentioned in Table 1 and further by sequencing. Confirmed clones were cultured in Luria Bertani (LB) media (HiMedia Laboratories, Mumbai, India) with 50 μg/mL kanamycin (AppliChem, Dermstadt, Germany) overnight at 37°C and the plasmids were isolated and transformed into Agrobacterium tumefaciens GV3101 strain by electroporation.
6
Serratia marcescens Molecular Characterization
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Serratia marcescens strain was purchased from MTCC, Chandigarh with the strain number MTCC 8708. Taq DNA polymerase master mix RED was purchased from Ampliqon, Phusion High-Fidelity DNA Polymerase mix, Restriction enzymes, T4 DNA ligase was purchased from New England Biolabs (NEB).
Luria-Bertani (LB) media, LB broth, Ampicillin, Kanamycin, Streptomycin Ni-NTA resin with the column was purchased from Hi-Media. IPTG was purchased from SRL chemicals. Primers are ordered from Shrimpex Biotech service Pvt. Ltd. PCR clean up kit were purchased from Smart prime ltd. Huminsulin 30/70 40 IU/mL cartridge was purchased from Eli Lilly Pvt Ltd.
Luria-Bertani (LB) media, LB broth, Ampicillin, Kanamycin, Streptomycin Ni-NTA resin with the column was purchased from Hi-Media. IPTG was purchased from SRL chemicals. Primers are ordered from Shrimpex Biotech service Pvt. Ltd. PCR clean up kit were purchased from Smart prime ltd. Huminsulin 30/70 40 IU/mL cartridge was purchased from Eli Lilly Pvt Ltd.
7
Overexpression and Complementation of E. coli Strains
8
Bacterial Strain Cultivation and Plasmid Expression
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A list of strains and plasmids used in this study is mentioned in Table S3. E. coli strains DH5α and BL21 (DE3) were grown in Luria-Bertani (LB) media (HiMedia Laboratories) at 37°C at 180 rpm. MS_RHII-RSD and Rel Msm -NTD were overexpressed using pET-21b plasmids.
For complementation studies, an E. coli GJ 12055 was used. E. coli GJ 12055 and complemented strains were grown at 30°C or 42°C in LB media containing 50 µg ml -1 kanamycin (Sigma). All E. coli strains harboring pET-21b or pSK760 plasmids were grown in the presence of 100 µg ml -1 ampicillin (Sigma). All strains were grown on plates containing LB media and 1.5% (w/v) agar (HiMedia Laboratories) in the presence of required antibiotics. M. smegmatis wild type mc2155 (WT) and ∆ms_rhII-rsd strains were grown in Middlebrook 7H9 broth (MB7H9, Difco) containing 2% (w/v) glucose (HiMedia Laboratories) and 0.05 % (v/v) Tween-80 (Sigma), at 37°C with agitation at 180 rpm. MB7H9 plates contained 1.5% agar and lacked Tween-80. Apramycin (Sigma) was used at a concentration of 50 µg ml -1 for ∆ms_rhII-rsd.
For complementation studies, an E. coli GJ 12055 was used. E. coli GJ 12055 and complemented strains were grown at 30°C or 42°C in LB media containing 50 µg ml -1 kanamycin (Sigma). All E. coli strains harboring pET-21b or pSK760 plasmids were grown in the presence of 100 µg ml -1 ampicillin (Sigma). All strains were grown on plates containing LB media and 1.5% (w/v) agar (HiMedia Laboratories) in the presence of required antibiotics. M. smegmatis wild type mc2155 (WT) and ∆ms_rhII-rsd strains were grown in Middlebrook 7H9 broth (MB7H9, Difco) containing 2% (w/v) glucose (HiMedia Laboratories) and 0.05 % (v/v) Tween-80 (Sigma), at 37°C with agitation at 180 rpm. MB7H9 plates contained 1.5% agar and lacked Tween-80. Apramycin (Sigma) was used at a concentration of 50 µg ml -1 for ∆ms_rhII-rsd.
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