The encoding region (5′-TGCGCATCCCCATCTGGCGCCCTTCGTTGTTGC-3′) of PI was synthesized with the addition of a BamHI site upstream and an EcoRI site downstream by Takara Bio Inc. (Otsu, Japan). The synthesized product was ligated into the pGEX-2T plasmid at the BamHI and EcoRI sites. The insert, cut with the restriction enzymes (Thermo Fisher Scientific), was subject to SDS-polyacrylamide gel electrophoresis (PAGE) and was identified by DNA sequence analysis. The Escherichia coli strain BL21 (DE3) was transformed with the recombinant plasmid pGEX-2T-PI or vector alone using the CaCl2 transformation method (24 (link)) and then grown in a lysogeny broth solution (Sigma-Aldrich) containing 100 mg/ml of ampicillin (Amresco) at 37°C. Isopropyl-β-D-thiogalactopyranoside (IPTG; Amresco), an inducer of β-galactosidase activity in bacteria, was added to a final concentration of 1 mM, and the solution was incubated at 37°C for 1–6 h. Following IPTG induction, bacterial pellets were obtained by centrifugation at 2,000 × g at 4°C.
Isopropyl β d thiogalactopyranoside iptg
Manufactured by Avantor
Sourced in United States, China
Isopropyl-β-D-thiogalactopyranoside (IPTG) is a synthetic molecular biology reagent commonly used in the regulation of gene expression in bacterial systems. It functions as an inducer, triggering the expression of genes under the control of the lac operon.
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Lab products found in correlation
Ampicillin, by Avantor (2 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Bl21 codonplus de3 ril, by Agilent Technologies (1 mentions)
Ni nta affinity column, by Qiagen (1 mentions)
Anti polyhistidine, by Merck Group (1 mentions)
Anti mouse igg conjugated to alkaline phosphatase, by Merck Group (1 mentions)
Protein marker, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Dna marker, by Takara Bio (1 mentions)
4 nitrophenyl palmitate p npp, by Merck Group (1 mentions)
Chloromycetin, by Avantor (1 mentions)
Kanamycin, by Avantor (1 mentions)
La taq polymerase, by Takara Bio (1 mentions)
Plasmid mini prepare kit, by Corning (1 mentions)
Dna mini kit, by Corning (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
6 protocols using isopropyl β d thiogalactopyranoside iptg
1
Plasmid Construction and Expression of PI Protein
2
Recombinant GBAA0190 Protein Expression
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The GBAA0190 gene was PCR-amplified from B. anthracis ATCC 14578 genomic DNA using specific primers with Nde I and Xho I restriction sites. The primers were as follows: forward primer 5′-GGCATATGAGAACTCTACTATCA-3′ and reverse primer 5′-GGCTCGAGTTATTTTATTTCTCTTTTATATTC-3′. Then the PCR products were cloned into pET19b vector (Novagen, Madison, WI, USA). For recombinant protein expression, the cloned DNA was transformed into E. coli strain BL21-CodonPlus (DE3) RIL (Agilent Technologies, Santa Clara, Ca, USA). Expression of r0190 was induced in Luria-Bertani broth in the presence of 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Amresco, Solon, OH, USA) for 12 h at 28 °C. The r0190 protein was purified by Fast Protein Liquid Chromatography (FPLC) using Ni-NTA affinity columns (Qiagen, Hilden, Germany). For production of immune serum against r0190, BALB/c mice were immunized 3 times every 2 weeks with 20 μg of purified r0190 protein. Two weeks after last immunization, blood samples from immunized mice were collected and used for the experiments including Western blot and immunoelectron microscopy.
3
Recombinant VP3 Protein Expression
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The full-length VP3 and its fragments were expressed in E. coli BL21 (DE3) Star (Novagen) with a 6×His tag at the N-terminus after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Amresco). Recombinant VP3 (rVP3, 37 kDa) was expressed at 37 °C for 4 h and solubilized from inclusion bodies with 2 M urea. Larger and shorter VP3 fragments composed of 130 and 50 amino acids, respectively, were expressed at 20 °C for 18 h. The shorter VP3 fragments were tagged to the C-terminus of GFP generating approximately 37 kDa proteins. All fragments could be recovered from the soluble fraction.
The rVP3 and its fragments were purified by affinity chromatography in a Ni-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) and were eluted with 500 mM imidazole. SDS-PAGE and Western blot were performed to evaluate the steps of the expression and purification procedures using a monoclonal antibody anti-polyhistidine as the primary antibody (Sigma) and anti-mouse IgG conjugated to alkaline phosphatase as the secondary antibody (Sigma). The Western blot detection was performed with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Promega).
The rVP3 and its fragments were purified by affinity chromatography in a Ni-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) and were eluted with 500 mM imidazole. SDS-PAGE and Western blot were performed to evaluate the steps of the expression and purification procedures using a monoclonal antibody anti-polyhistidine as the primary antibody (Sigma) and anti-mouse IgG conjugated to alkaline phosphatase as the secondary antibody (Sigma). The Western blot detection was performed with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Promega).
4
Lipase Activity Screening and Characterization
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LATaq polymerase, T4 DNA ligase, DNA marker and restriction enzymes were purchased from TaKaRa Biotechnology (Otsu, Japan). Protein marker was purchased from MBI Fermentas (Vilnius, Lithuania). Isopropyl-β -Dthiogalactopyranoside (IPTG), ampicillin, kanamycin and chloromycetin were purchased from Amresco (Shanghai Genebase, China). DNA Mini kit and Plasmid Mini Prepare kit were purchased from Axygen Biosciences (Union City, CA, USA). The substrate 4-nitrophenyl palmitate (pNPP) was purchased from Sigma. Other chemicals were obtained commercially and were of reagent grade.
Luria Bertani medium was used for E. coli and Bacillus spp. propagation with appropriate resistance. Rhodamine B agar plates (yeast extract 0.5 %, tryptone 1 %, NaCl 1 %, olive oil 1 %, rhodamine B 0.001 % and agar 1.5 %) were used for strain screening. Lipase basal medium (olive oil 1 %, tryptone 0.5 %, yeast extract 0.25 %, KH 2 PO 4 0.07 %, K 2 HPO 4 0.03 %) was used for wild-strain lipase activity analysis. kanamycin was used at 50 μg/ml, chloromycetin was used at 10 μg/ml, and ampicillin was used at 100 μg/ml.
Luria Bertani medium was used for E. coli and Bacillus spp. propagation with appropriate resistance. Rhodamine B agar plates (yeast extract 0.5 %, tryptone 1 %, NaCl 1 %, olive oil 1 %, rhodamine B 0.001 % and agar 1.5 %) were used for strain screening. Lipase basal medium (olive oil 1 %, tryptone 0.5 %, yeast extract 0.25 %, KH 2 PO 4 0.07 %, K 2 HPO 4 0.03 %) was used for wild-strain lipase activity analysis. kanamycin was used at 50 μg/ml, chloromycetin was used at 10 μg/ml, and ampicillin was used at 100 μg/ml.
5
Bacterial Strain Cultivation and Maintenance
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The bacterial strains used in this study are listed in Table D in S1 Text and details of their construction are outlined in the supplementary materials and methods (S1 Text ). Plasmids and primers used in this study are listed in Tables E and F in S1 Text , respectively. S. aureus strains were grown at 37°C in Tryptic soy broth medium (TSB; Difco) or on Tryptic soy agar (TSA; Difco) supplemented with appropriate antibiotics when required (erythromycin 10 μg/ml, chloramphenicol 10 μg/ml or tetracycline 5 μg/ml; Sigma-Aldrich) or with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG; VWR). For growth studies in minimal media cells were grown in SSM9PR minimal media containing 1 x M9 salts, 2 mM MgSO4, 0.1 mM CaCl2, 1% glucose, 1% casaminoacids, 1 mM Thiamine-HCl and 0.05 mM nicotinamide at 37°C. E. coli strains were grown at 30°C or 37°C in Luria-Bertani broth medium (LB broth; Difco), on LB agar (Difco), supplemented with 100 μg/ml ampicillin (Sigma-Aldrich) or 50 μg/ml kanamycin (Sigma-Aldrich), 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; VWR) and 0.5 mM IPTG when required.
6
Rv0158 Protein Overexpression and Purification from Mycobacterium tuberculosis
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The nucleotide sequence of rv0158 ORF was PCR-amplified from Mtb H37Rv genome and cloned into pet28a plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) cells. At OD600 nm of ∼0.6, 0.5 mM isopropyl-β-D thiogalactopyranoside (IPTG; VWR Chemicals, cat. no.- 0487–10 g) were added to induce expression of His-tagged Rv0158 for 24 hr at 18 °C. The culture medium containing the bacteria was centrifuged at 3000 g for 10 min, and the cell pellet was suspended in lysis buffer 25 mM Tris-Cl pH 8.0, 500 mM NaCl, 5% glycerol, 5 mM βmercaptoethanol, 5 mM imidazole, and 2 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Aldrich, cat. no.- P9625-14). The cells were sonicated for 30 min on ice, followed by centrifugation at 3000 g for 45 min. His-tagged Rv0158 protein in the supernatant was purified on a Ni-NTA (Qiagen, cat. no.- 30210) resin chromatography column and eluted with elution buffer 25 mM Tris-Cl pH 8.0, 500 mM NaCl, 5% glycerol, 5 mM βmercaptoethanol, 250 mM imidazole and 2 mM phenylmethylsulfonyl fluoride (PMSF), followed by dialysis to remove imidazole. The protein purity was evaluated by SDS-PAGE and quantified by BCA assay (Thermo Scientific, cat. no. 23225, Rockford, IL).
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