Bl21 de3

Manufactured by Merck Group

Sourced in United States, Germany

BL21 (DE3) is a Escherichia coli bacterial strain commonly used in molecular biology laboratories for the expression of recombinant proteins. It is designed to efficiently express proteins under the control of the T7 promoter system.

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Lab products found in correlation

Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (4 mentions) Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Yeast extract, by Merck Group (3 mentions) Kanamycin, by Merck Group (2 mentions) Anti his, by Merck Group (2 mentions) Ptrchis topo, by Thermo Fisher Scientific (2 mentions) Pet32a, by Merck Group (2 mentions) Endotoxinout resin, by G Biosciences (2 mentions) Bradford reagent, by Bio-Rad (2 mentions) Glycerol, by Merck Group (2 mentions) Lactose, by Merck Group (2 mentions) Tryptone, by Merck Group (2 mentions) Bhi broth, by Thermo Fisher Scientific (2 mentions) Er2566, by New England Biolabs (2 mentions) Xl1 blue, by Agilent Technologies (1 mentions) By4741, by Thermo Fisher Scientific (1 mentions) By4742, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by AppliChem (1 mentions) Alexa fluor 546, by Thermo Fisher Scientific (1 mentions) Histraptm ff column, by GE Healthcare (1 mentions)

52 protocols using bl21 de3

Bacterial Strain Cultivation and Plasmid Selection

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Escherichia coli DH5α (Millipore Corp., Burlington, MA, USA), BL21 (DE3) (Millipore Corp., Burlington, MA, USA), and XL-1 Blue (Agilent Technologies, Waldbronn, Germany) strains were used in this study. The shake flask fermentation was performed in LB medium containing 50 μg/mL kanamycin, 100 μg/mL ampicillin, or 34 μg/mL chloramphenicol for plasmid selection and maintenance.
All chemicals used in this study were reagent grade, and purchased from Aladdin Industrial Inc. (Shanghai, China) or Sangon Biotech (Shanghai, China). All enzymes for molecular biology experiments were purchased from TaKaRa Biotech (Dalian, China), New England Biolabs (Ipswich, MA, USA), or Vazyme Biotech (Nanjing, China).

Luo H., He W., Dai Z., Zhang Z., Bao Y., Li D, & Zhu P. (2022). Concurrent Production of α- and β-Carotenes with Different Stoichiometries Displaying Diverse Antioxidative Activities via Lycopene Cyclases-Based Rational System. Antioxidants, 11(11), 2267.

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Bacterial Growth and Cloning Protocol

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Bacteria were grown overnight (O/N) in Lysogeny Broth (LB) or LB-agar plates at 30 °C for Bacillus spp. and at 37 °C for Escherichia coli and Staphylococcus epidermidis. Listeria ivanovii was grown at 37 °C in brain heart infusion (BHI). E. coli 10-beta and BL21(DE3) (Millipore corporation, Burlington, MA, USA) competent cells were used for cloning and expression experiments, respectively. E. coli cells containing the pET30a vector were selected in LB medium containing 50 µg/mL of kanamycin (Sigma, Saint-Louis, MO, USA) at 37 °C, unless otherwise indicated.

Leprince A., Nuytten M., Gillis A, & Mahillon J. (2020). Characterization of PlyB221 and PlyP32, Two Novel Endolysins Encoded by Phages Preying on the Bacillus cereus Group. Viruses, 12(9), 1052.

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Screening for DNA Suppressors of NEDD4w4

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E. coli strain DH5 was used for plasmid propagation and BL21(DE3) (Millipore) for protein production. The yeast Saccharomyces cerevisiae strains used in this study are: INV (Invitrogen), PJ69-4A (James et al., 1996) , MHY501 (Chen et al., 1993) , YYM4 (Stawiecka-Mirota et al., 2008) , BY4741, BY4742, BY4741 atg2  BY4741 atg13 , BY4741 atg14 , BY4741 atg18 (Open Biosystems), BY4741 LAS17-GFP (Invitrogen), CRY2040 HSP104-yTagRFP-T and CRY2041 HSP42-yTagRFP-T (Malcova et al., 2016) .
Yeast media used were YPD (1% yeast extract, 1% peptone, 2% glucose), SC synthetic complete medium (0.67% yeast nitrogen base without amino acids, 2% glucose) with desired supplements (adenine, uracil, amino acids (aa)); SCgal contains 2% galactose, and SCraf contains 2% raffinose instead of glucose.
To compare the growth of strains, middle-log phase cultures were diluted with water to have 1 OD 600 equivalents/ml. Aliquots of five-fold serial dilutions of these cell suspensions were spotted onto the medium and incubated as indicated.
Multicopy suppressor screen for DNA fragments restoring growth of yeast cells bearing pYES-NEDD4w4 (with URA3 marker) (Gajewska et al., 2003) was performed using a yeast genomic library from ura3 strain on the YEp351 multicopy plasmid (gift of M. Wysocka-Kapcinska, IBB PAS).

Kaminska J., Rzepnikowska W., Polak A., Flis K., Soczewka P., Bala K., Sienko M., Grynberg M., Kaliszewski P., Urbanek A., Ayscough K, & Zoladek T. (2016). Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress. The international journal of biochemistry & cell biology, 79.

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Expression and Purification of Truncated Tau Protein

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Human truncated tau151-391 (numbering according to the longest human tau isoform Tau40) was expressed in Escherichia coli strain BL21(DE3) (Sigma-Aldrich, St. Louise, Missouri, United States) from a pET-17 expression vector and purified from bacterial lysates as described previously [39 (link)], except that the phosphocellulose step was omitted and size-exclusion chromatography was performed in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) (AppliChem GmbH, Darmstadt, Germany). Purified tau protein was stored in PBS in working aliquots at −70°C. The purity of tau protein was subsequently verified by gradient SDS gel electrophoresis (5 to 20% gel), Coomassie blue staining and Western blot analysis with DC25 antibody (AXON Neuroscience SE (Bratislava, Slovakia), recognizes residues 347–354 of the longest human tau isoform Tau40). The fluorescently tagged tau protein was prepared by labelling with Alexa Fluor 546 (Invitrogen, Carlsbad, California, United States) according to the manufacturer’s recommendations.

Majerova P., Zilkova M., Kazmerova Z., Kovac A., Paholikova K., Kovacech B., Zilka N, & Novak M. (2014). Microglia display modest phagocytic capacity for extracellular tau oligomers. Journal of Neuroinflammation, 11, 161.

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Recombinant Ts-MAPRC2 Protein Expression

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The restriction digestion of the plasmid (pMD19-T/ Ts-MAPRC2) was carried out using the enzymes viz., EcoR I and Hind III was cloned into the prokaryotic expression vector pET-32a (+) (Novagen, Beijing, China). Recombinant plasmid (pET32a (+)/Ts-MAPRC2) was then processed into BL21 (DE3) and induced protein expression by 1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich, Shanghai, China). Further, cells were harvested and lysed by lysozyme (10 μg/mL) (Sigma-Aldrich, Kenilworth, NJ, USA), and by sonication. The sonicated outputs were subsequently confirmed on the SDS-PAGE (12% w/v). Recombinant Ts-MAPRC2 (rTs-MAPRC2) protein was purified by the His-TrapTM FF column following the manufacturer’s instructions (GE Healthcare, Piscataway, NJ, USA), and protein concentration was calculated by Pierce-TM BCA-Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Empty pET32a (histidine-tagged) protein was purified and expressed using the same procedure mentioned above for Ts-MAPRC2 and utilized vector-protein as a negative control. Pictures of the SDS-PAGE carrying the rTs-MAPRC2 purified protein were taken. The stock of rTs-MAPRC2 protein was prepared and preserved at −80 °C until the next experiments.

Aleem M.T., Shi J., Yu Z., Wen Z., Zhang Y., Liang M., Lakho S.A., Haseeb M., Ali H., Hassan M.W., Song X., Li X., Xu L, & Yan R. (2021). Characterization of Membrane-Associated Progesterone Receptor Component-2 (MAPRC2) from Trichinella spiralis and Its Interaction with Progesterone and Mifepristone. Vaccines, 9(8), 934.

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Overexpression and Nickel Sensitivity Assay

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BL21(DE3) (Sigma, CMC0014) was transformed with His-TmCorA, MRS2(71-443)-FLAG, its mutants or without insert in a pET28a backbone. The transformants were inoculated and grown in LBK medium, shaking at 250 rpm and 37°C overnight. The overnight cultures were used to inoculate fresh LBK in a 1:100 ratio and grown until they reached an OD600 of 0.6. The bacterial cultures were then diluted 10-fold serially with LBK medium, spotted onto LBK agar plates containing 0.1 mM IPTG, and 0 or 1.8 mM NiCl₂, and incubated at 30°C overnight. Plates were imaged using ChemiDoc MP Imaging System (Bio-rad).

Lai L.T., Balaraman J., Zhou F, & Matthies D. (2023). Cryo-EM structures of human magnesium channel MRS2 reveal gating and regulatory mechanisms. bioRxiv.

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Biosynthesis of Hydroxytyrosol via Engineered E. coli

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Restriction enzymes, DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase were purchased from Takara Bio, Inc. (Dalian, China). Hieff Clone^TM Plus Multi One Step Cloning Kit was purchased from YEASEN Biotechnology Co., Ltd. (Shanghai, China). Oligonucleotides were purchased from Life Technologies (Shanghai, China). 4-Hydroxyphenylacetate, tyrosine, tyrosol, hydroxytyrosol, tyramine, dopamine, 3,4-DHPAA, 4HPAA, sodium periodate, dansylcholoride, and NADH were all purchased from Sigma-Aldrich (St. Louis, USA).
Escherichia coli strain MC1061, BL21(DE3), and BW25113 were used for cloning, protein overexpression, and hydroxytyrosol biosynthesis, respectively. Strains were grown at 37 °C. Kanamycin (50 μg/mL) were supplemented when necessary. Hydroxytyrosol biosynthesis was performed in yeast extract M9Y medium (M9 minimal salts (Becton, Dickinson and Company), 1% (w/v) glucose, 5 mM MgSO₄, 0.1 mM CaCl₂ supplemented with 0.025% (w/v) of yeast extract)^{14 (link)}, while other cultures were done in Luria-Bertani (LB) medium if not specially indicated.

Chen W., Yao J., Meng J., Han W., Tao Y., Chen Y., Guo Y., Shi G., He Y., Jin J.M, & Tang S.Y. (2019). Promiscuous enzymatic activity-aided multiple-pathway network design for metabolic flux rearrangement in hydroxytyrosol biosynthesis. Nature Communications, 10, 960.

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Purification of Zinc Finger Proteins

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The HBG-4kb ZF and control ZF were expressed in and purified from E. coli as described previously with minor modifications (26 (link), 29 (link)). BL21(DE3) (Sigma) competent cells harboring the ZF-DBD expression plasmids were incubated with 1 mmol/liter IPTG (isopropyl-β-d-1-thiogalactopyranoside; Sigma) at 37°C for 4 h. The cells were lysed using a French press with the high-power mode for 3 cycles. The purification of the ZF-DBDs was done with HisLink resin-packed columns (Promega, Madison, WI) as previously described (29 (link)). The supernatant of the cell lysate was passed through the resin column and washed with 5 column volumes of wash buffer (100 mmol/liter HEPES, 10 mmol/liter imidazole, pH 7.5) and eluted with 5 column volumes of elution buffer (100 mmol/liter HEPES, 1 mol/liter imidazole, pH 7.5). Desalting and concentration of the purified ZF-DBDs were done using 3-kDa-cutoff Vivaspin columns (GE Healthcare, Pittsburgh, PA) following the manufacturer's instructions. The concentration of purified ZF-DBDs was determined using bovine serum albumin (BSA) standards (NEB, Ipswich, MA).

Shen Y., Bassett M.A., Gurumurthy A., Nar R., Knudson I.J., Guy C.R., Perez A., Mellen R.W., Ikeda M., Hossain M.A., Huang S., Igarashi K, & Bungert J. (2018). Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression. Molecular and Cellular Biology, 38(19), e00197-18.

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Recombinant Galectin-9 Protein Purification

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Human full-length Gal-9, mouse full-length Gal-9, and GST-fused human N-CRD or C-CRD Gal-9 were expressed in Escherichia coli and purified as described previously (6 (link)). In brief, pET21a-Gal-9 construct was transformed into E. coli strain BL21 (DE3) (Sigma–Aldrich). Cells were allowed to grow in 2xYT medium to log phase, and then IPTG was added to a final concentration of 0.1 mM. The culture was incubated at 20 °C overnight and then harvested and lysed in B-PER reagent (Thermo Fisher Scientific) per the manufacturer's instruction. Gal-9 was then purified by affinity chromatography on a lactosyl-agarose column (Sigma–Aldrich). For the purification of GST-fused N-CRD or C-CRD Gal-9, pGEX-N-CRD or pGEX-C-CRD constructs were expressed in E. coli strain BL21 and then purified by affinity chromatography with glutathione-conjugated Sepharose beads (GenScript) according to the manufacturer’s instruction.

Yang R., Sun L., Li C.F., Wang Y.H., Xia W., Liu B., Chu Y.Y., Bover L., Vien L, & Hung M.C. (2022). Development and characterization of anti-galectin-9 antibodies that protect T cells from galectin-9-induced cell death. The Journal of Biological Chemistry, 298(4), 101821.

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Bacterial Expression of Recombinant UcMTs

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The recombinant expression vectors were transformed in competent E. coli strain BL21 (DE3) (Sigma-Aldrich, Saint Louis, MO, USA). To this end, 200 µL of competent cells BL21 (DE3) were incubated with 50 ng of recombinant expression vector containing UcMTs-GST. Then, 800 µL of SOC medium (2% (w/v) tryptone, 0.5% (w/v) of yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mm MgSO₄, and 20 mM glucose) were added, and cultivated at 37 °C for 45 min. An aliquot of 200 µL was cultured on solid LB medium containing 100 µg L⁻¹ of carbenicillin in a Petri dish overnight. Individual colonies were selected for each UcMT, cultivated with LB medium, and stored at −80 °C in LB medium containing 15% glycerol.

Zúñiga A., Laporte D., González A., Gómez M., Sáez C.A, & Moenne A. (2019). Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta). International Journal of Molecular Sciences, 21(1), 153.

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