MTB H37Rv standard strain, Escherichia coli dh5a, BL21 (DE3), and pet30a vectors were preserved in our laboratory; PMD18-T vector was purchased from Bao Bioengieering Co., Ltd (Dalian, China); instrumental enzymes and reagents: restriction endonuclease, T4 ligase, tap enzyme, isopropyl β-d-1-thiogalactopyranoside (IPTG), and purification assay were purchased from Takara Bio (Mountain View, CA, USA); HIS-tag purification kit was purchased from Novagen (Madison, WI, USA); horseradish peroxidase (HRP) goat anti-human immunoglobulin G (IgG) was from Jackson ImmunoResearch (West Grove, PA, USA); and primers were synthesized by a Shanghai bioengineering company and sequenced by a Yingjun bioengineering company.
Isopropyl β d 1 thiogalactopyranoside (iptg)
Manufactured by Takara Bio
Sourced in Japan, China
IPTG is a synthetic analogue of lactose that is commonly used as an inducer in bacterial expression systems. It functions by binding to the lac repressor protein, thereby relieving repression of the lac operon and allowing transcription of the target gene.
Automatically generated - may contain errors
Lab products found in correlation
Restriction endonuclease, by Takara Bio (3 mentions)
Nickel nta agarose, by Qiagen (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Ex taq, by Takara Bio (2 mentions)
Dna polymerase, by Takara Bio (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Ni nta agarose chromatography, by Merck Group (2 mentions)
Ampicillin, by Takara Bio (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
T4 ligase, by Takara Bio (1 mentions)
Chondroitin 4 sulfate, by Merck Group (1 mentions)
Potassium dihydrogen phosphate kh2po4, by Merck Group (1 mentions)
Mighty ta cloning kit, by Takara Bio (1 mentions)
Ni nitrilotriacetic acid nta agarose, by Qiagen (1 mentions)
Mighty mix, by Takara Bio (1 mentions)
Can get signal, by Toyobo (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Lb broth, by Merck Group (1 mentions)
23 protocols using isopropyl β d 1 thiogalactopyranoside (iptg)
1
Recombinant Protein Expression Workflow
2
Molecular Chaperones Enhance Solvent Resistance
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Molecular chaperones were overexpressed to evaluate their role in resistance against organic solvents. Thirty molecular chaperones coding genes (nlpE, hybE, ravA, ycaL, clpA, clpX, cbpA, hscC, hslO, ibpA, ibpB, nfuA, ppiD, skp, secB, surA, ycdY, yegD, yrhB, clpB, hchA, grpE, htpG, groL, lolA, djlA, bepA, yajL, dnaK and dnaJ) were selected and cloned with genomic DNA of E. coli JM109 and primers as listed in Additional file 1 : Table S1. All the PCR products were ligated into pQE80L digested with BamHI and HindIII (Takara Ltd Ltd, Shanghai) using Exanse II (Vazyme Ltd, Nanjing). The recombinant plasmids were transformed into E. coli JM109 to form the engineered strains. All the chaperones were verified by digestion with BamHI and sequencing.
Each colony of engineered E. coli JM109 was picked up and inoculated into LB medium and cultivated at 37 °C and 180 rpm overnight. Then, 1% (v/v) culture was transferred into 30-mL fresh LB medium (10 g L−1 tryptone, 5 g L−1 yeast extract, 10 g L−1 NaCl, pH 7.0) and further cultivated at 37 °C and 180 rpm. When the OD600 reached to 0.3, 0.2-mM isopropyl β-d -1-thiogalactopyranoside (IPTG, Takara) was added and incubated at 30 °C and 180 rpm for 6 h. Then, the cells were collected via centrifugation at 8800×g and 4 °C, and disrupted using high-pressure homogenizer (ATS BASIC-II, Shanghai). Chaperones were verified by SDS-PAGE.
Each colony of engineered E. coli JM109 was picked up and inoculated into LB medium and cultivated at 37 °C and 180 rpm overnight. Then, 1% (v/v) culture was transferred into 30-mL fresh LB medium (10 g L−1 tryptone, 5 g L−1 yeast extract, 10 g L−1 NaCl, pH 7.0) and further cultivated at 37 °C and 180 rpm. When the OD600 reached to 0.3, 0.2-mM isopropyl β-
3
Nanohydroxyapatite Synthesis Protocol
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Chemicals including chondroitin 4-sulfate, potassium dihydrogen phosphate (KH2PO4), and kanamycin were all purchased from Sigma Aldrich; (USA), and nickel NTA agarose was purchased from Qiagen (USA). Isopropyl β-d -1-thiogalactopyranoside (IPTG) was prepared from Takara (Japan). The nano-hydroxyapatite powder was obtained from Fine Nano company (Iran, Tehran). Deionized water and phosphate buffer were used to make the solutions.
4
Ligand Capture Assay for GI.1 HuNoV
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The BSDS was used to capture the potential ligands of GI.1 HuNoV (Figure 1 ). The P protein of GI.1 HuNoV was anchored on the surface of E. coli BL21(DE3) with the N terminal (InaQn) of the ice nucleoprotein of Pseudomonas syringae. At the same time, an amino acid sequence that could be recognized by thrombin (TB) was added between the anchor protein and the P protein of GI.1 HuNoV. Therefore, the P protein could be separated from the surface of the host bacteria by thrombin digestion, to pull down the protein ligands of GI.1 HuNoV from the oyster tissue. E. coli BL21(DE3) with pET28a-inaQn-TB-I.1p (P) was constructed as described earlier [23 (link)]. The E. coli BL21(DE3) with pET28a-inaQn-TB-II.4p was used as the positive control, and the E. coli BL21(DE3) with pET28a-inaQn-TB (T) was constructed as the negative control. The recombinant E. coli BL21(DE3) was cultured in Luria–Bertani (LB) liquid medium (Huankai Microbial Co., Ltd., Guangzhou, China) containing 100.0 μg/mL kanamycin at 37 °C and shaken (150 rpm) until OD600 reached ~0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG; Takara Bio Inc., Beijing, China) was added to a final concentration of 0.5 mM and shaken (150 rpm) for 12 h at 25 °C. The cells were then rinsed with PBS and stored at 4 °C for further use.
5
Genetic Manipulation of E. coli Strains
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RAW264.2 cells were purchased from ATCC. All plasmids and bacterial strains used in this study are shown in Tables 2 to 4 . E. coli K-12 BW25113 (wild type [WT]) and single gene-deleted mutant strains (ΔompA: opmA::kan for JW0940-KC, ΔfepA: fepA::kan for JW5086-KC, ΔcirA: cirA::kan for JW2142-KC, ΔdegP: degP::kan for JW0157-KC, and ΔompC: ompC::kan for JW2203-KC) were provided by National BioResource Project (NIG, Japan). The competent cell BL21(DE3) strain and DH5α strain were purchased from TaKaRa (Shiga, Japan). LB broth was purchased from Sigma/Merck (Tokyo, Japan). The plasmid pET22b(+) was purchased from Novagen/Merck (Tokyo, Japan). pTV118N vector was gifted by Ishijima (Kyoto Prefectural University). Ex Taq, Mighty TA-cloning kit containing pMD T vector, DNA ligation kit Mighty Mix, in-fusion HD cloning kit, isopropyl β-d -1-thiogalactopyranoside (IPTG), and TB Green premix Ex Taq II were purchased from TaKaRa (Shiga, Japan). Ni-nitrilotriacetic acid (NTA) agarose was purchased from Qiagen (Hilden, Germany). cOmplete Mini protease inhibitor was purchased from Roche/Nippon Gene (Tokyo, Japan). Anti-OmpC antibody was purchased from MyBioSource (Vancouver, Canada). Isogen II was purchased from Nippon Gene (Tokyo, Japan). Can Get Signal and ReverTra Ace quantitative PCR (qPCR) reverse transcription (RT) kit were purchased from Toyobo (Osaka, Japan).
6
Cloning and Protein Expression in E. coli
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Escherichia coli (E. coli) Top10 and plasmid pET-28a (+) (Invitrogen, Shanghai, China) were used as cloning host and vector in this study. E. coli Shuffle T7 express competent cells (Invitrogen, Shanghai China). A SanPrep Column plasmid mini-preps kits were purchased from Sangon Biotech (Shanghai, China) to extract plasmid. Bradford protein assay kit (Sangon Shanghai, China) was used for measurement of protein concentration. The Molecular protein marker (code No: 3595Q) was purchased from TaKaRa Biotechnology Co. Ltd. Dalian, China. Isopropyl β-d -1-thiogalactopyranoside (IPTG) (TaKaRa Biotechnology Co. Ltd. Dalian, China). Lecithin (Soybean PC > 98%), CDCl3, Methanol (Aladdin Industrial corporation #1008 Qigang Rd, Nanqiao Town, Fengxian Shanghai (201406), China). Triphenyl Phosphate (TPP, purity 99.9%) (Shanghai Macklin Biochemical Co. Ltd., Shanghai, China). Other chemicals and reagents used in present study were of analytical grade.
7
Cloning and Expression of Alkalibacterium Xylanase
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The bacterial strain Alkalibacterium sp. SL3 used for obtaining the xylanase gene was isolated from a soda lake during a previous study (Wang et al., 2016 (link)). E. coli Top 10 and the pMD 18-T vector (TaKaRa, Otsu, Japan) were used for gene cloning. Vector pET-22b(+; Novagen, San Diego, CA, USA) and E. coli BL21 (DE3; TaKaRa, Otsu, Japan) were used for gene expression. Kits for genomic DNA isolation, DNA purification, and plasmid isolation were purchased from Omega (Norcross, GA, USA). The Genome Walking kit, restriction endonucleases, T4 DNA ligase, DNA polymerase, dNTPs, and Isopropyl-β-D-1-thiogalactopyranoside (IPTG) were purchased from TaKaRa (Otsu, Japan). Nickel-NTA agarose (Qiagen, Valencia, CA, USA) was used to purify the His6-tagged protein. The substrate beechwood xylan was purchased from Sigma (St. Louis, MO, USA). Corncob xylan was purchased from Yuanye (Shanghai, China) All other chemicals were of analytical grade and commercially available.
8
Protein-Protein Interaction Assay
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RGA-GST and ABI4-MBP fusion proteins were induced by 0.4 mM isopropyl β-D-1-thiogalactopyranoside (9030, Takara) and expressed in E. coli cells. The recombinant proteins were purified using GST-bind resin (A10018S, Abmart) and amylose resin (E8021S, NEB), respectively. The purified recombinant bait, prey proteins, and GST-bind or amylose resin were incubated in binding buffer containing 10 mM Tris-HCl (pH 7.5) (0497, Amresco), 1 mM EDTA (0322-1KG, Amresco), 1 mM EGTA (324626, Sigma-Aldrich), 0.5% Nonidet P-40, 1% Triton X-100 (A600198, BBi), 150 mM NaCl (V900058, Sigma-Aldrich), and 1 mM DTT (20290, Pierce) at 4°C for 4 h. Then the bound proteins were eluted and separated by SDS-PAGE and evaluated by immunoblotting.
9
Purification of Recombinant Proteins from E. coli
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β-Cyclodextrin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroform was of HPLC grade from Kermel Chemical Reagent Co., Ltd. (Tianjin, China). Isopropyl β-d -1-thiogalactopyranoside (IPTG) was purchased from the TaKaRa Biotechnology Co. Ltd. (Dalian, China), Ni2+-NTA Sepharose fast flow, Sephadex G-25 Fine and DEAE Sepharose Fast Flow chromatographic packing were all purchased from GE Healthcare (Boston, MA, USA). E. coli Shuffle T7 Express Competent was purchased from New England BioLabs (Beijing, China). Bicinchoninic acid (BCA) Protein Assay Kit was from Sangon Biotech, Shanghai Co., Ltd. (Shanghai, China). Water was purified with Millipore (Bedford, MA, USA) Milli-Q water system. All other regents were of analytical grade.
10
Recombinant Mycobacterial Protein Production
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The regulatory genes were amplified by PCR using specific primers from genomic DNA of M. tuberculosis H37Rv and were cloned into pET28a to produce recombinant vectors (Table S1 and S2). E. coli BL21 cells, which were transformed with the recombinant plasmid, were grown in 200 ml of LB medium up to OD600 of 0.6. Protein expression was induced by the addition of 0.3 mM isopropyl β-D-1-thiogalactopyranoside (TaKaRa). Harvested cells were resuspended and sonicated in binding buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl; and 10 mM imidazole), and the lysate was centrifuged at 10,000 × g for 30 min. The cleared supernatant was loaded onto the affinity column. The column-bound protein was washed with buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl; and 40 mM imidazole). The protein was then eluted using elution buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl; and 250 mM imidazole). The elution was dialyzed overnight and stored at −80 °C. Protein concentration was detected with a Coomassie Brilliant Blue assay.
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