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E coli bl21 de3 cells

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E. coli BL21 (DE3) cells are a common laboratory strain of Escherichia coli bacteria. They are designed for the expression of recombinant proteins. The cells contain the DE3 lysogen, which allows for the inducible expression of target proteins under the control of the T7 promoter system.

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Lab products found in correlation

Histrap ff, by GE Healthcare (3 mentions) Superdex 200 16 600 column, by GE Healthcare (3 mentions) Pet28b vector, by Thermo Fisher Scientific (3 mentions) Sars cov 2 wuhan hu 1 envelope protein, by Genewiz (3 mentions) Champion pet sumo plasmid, by Thermo Fisher Scientific (3 mentions) Sanger sequencing, by Genewiz (3 mentions) Pd 10 column, by GE Healthcare (2 mentions) Ni nta, by Qiagen (2 mentions) Irdye 680rd goat anti mouse secondary antibody, by LI COR (2 mentions) Mouse gst, by BioLegend (2 mentions) Anti his monoclonal primary antibodies, by LI COR (2 mentions) T4 dna ligase, by New England Biolabs (2 mentions) Endotoxin e coli standards kits, by Lonza (2 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (2 mentions) Histrap column, by GE Healthcare (2 mentions) E coli dh5α, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Superdex 200 increase 10 300 gl, by GE Healthcare (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Organic acid, by Merck Group (1 mentions)

74 protocols using e coli bl21 de3 cells

1

Bacterial Strain and Plasmid Engineering

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All the bacterial strains, plasmids, and primers used in the current study are listed in Supplementary Tables S1-S3, respectively. The primers used for genetic manipulation were ordered from Integrated DNA Technologies (San Diego, CA, United States). DNA extraction and genetic engineering procedures were performed as described (Sambrook et al., 1989 (link)). For plasmid harboring and cloning, Escherichia coli XL1-Blue (Stratagene, La Jolla, CA, United States) was grown overnight (16 h) in Luria-Bertani, Lennox medium (LB-Lennox) at pH 7.5 under 37°C and 200 rpm. If required, ampicillin (100 μg/mL), chloramphenicol (50 μg/mL) and kanamycin (50 μg/mL) were introduced. Detailed plasmid construction strategies are described in the Supplementary Material. Positive constructed clones were transformed into the appropriate competent E. coli BL21(DE3) cells (Invitrogen, Carlsbad, CA, United States), and competent E. coli BL21(DE3) cells harboring plasmid pMCS69 for production of soluble proteins and PHA spheres, respectively. Plasmid pMCS69 allows the synthesis of the precursor R-3-hydroxybutryl-coenzyme A (CoA), which is essential to biosynthesis of PHA spheres.
Wong J.X., Gonzalez-Miro M., Sutherland-Smith A.J, & Rehm B.H. (2020). Covalent Functionalization of Bioengineered Polyhydroxyalkanoate Spheres Directed by Specific Protein-Protein Interactions. Frontiers in Bioengineering and Biotechnology, 8, 44.
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2

Recombinant Expression and Purification of SznF

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The sznF gene was amplified from the cosmid encoding the szn gene cluster using the primers in Supplementary Table 4. The amplified gene was digested with NdeI and HindIII and ligated into the pET28a vector as described above for pET28a-SznE. The plasmid was transformed into E. coli BL21 (DE3) cells (Invitrogen), and the protein was expressed and purified using the same procedure as described for SznE with the exception that the exchange buffer contained 20 mM MOPS pH 7.5 instead of 20 mM HEPES pH 8.
Ng T.L., Rohac R., Mitchell A., Boal A.K, & Balskus E.P. (2019). An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin. Nature, 566(7742), 94-99.
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3

Characterization of Multidrug-Resistant Acinetobacter Phage

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Bacterial strains were acquired either from the American Type Culture Collection (S. typhimurium ATCC 19585, P. aeruginosa ATCC 15692, and Klebsiella oxytoca ATCC 131821) or from the Spanish Type Culture Collection (Escherichia coli O157:H7 CECT 47821). Clinical Acinetobacter isolates are multi-resistant and were kindly provided by the Hospital of Braga with patterns of antibiotic resistance given for each strain. All strains were grown in Lysogeny Broth (LB; Liofilchem) at 37°C and 120 rpm. For transformation, chemically competent E. coli TOP10 and E. coli BL21(DE3) cells (Invitrogen) were prepared for cloning and recombinant protein expression respectively. The Acinetobacter phage vb_AbaP_CEB1 (encoding the ABgp46 endolysin) was isolated from waste effluents and belongs to the Centre of Biological Engineering phage collection (Braga, Portugal). This phage belongs to the Podoviridae family and is a T7 likeviruses EDTA was acquired from Pronalab while isopropyl-β-D-thiogalactopyranoside (IPTG), HEPES and the organic acids were purchased from Sigma-Aldrich.
Oliveira H., Vilas Boas D., Mesnage S., Kluskens L.D., Lavigne R., Sillankorva S., Secundo F, & Azeredo J. (2016). Structural and Enzymatic Characterization of ABgp46, a Novel Phage Endolysin with Broad Anti-Gram-Negative Bacterial Activity. Frontiers in Microbiology, 7, 208.
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4

Recombinant Protein Expression in E. coli

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All recombinant proteins were expressed in E. coli BL21(DE3) cells (Invitrogen), except for Prc mutant proteins, including PrcΔPDZ (Δ244-339), Prc-K477A, Prc-K308W, Prc-L245A, Prc-L340G/A, and Prc-L340G/L245A, which were expressed in the Δprc strain MR812. Cells were cultured to an optical density at 600 nm of 0.6–0.8 and induced with 1 mM isopropyl β-d-thiogalactopyranoside at 25 °C for 4 h. Cell pellets were suspended in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl and then ruptured by French press (Avestin). After centrifugation at 35,000 × g at 4 °C for 45 min, the supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) column and washed with 20 mM imidazole. The protein fraction eluted with 250 mM imidazole was purified further by MonoQ 5/50 GL column (GE Healthcare) at pH 8.0, and Superdex 200 10/300 GL column (GE Healthcare) was equilibrated in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl for Prc and sNlpI, but in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 1 mM DTT for sMepS. Protein purity was analyzed by SDS-PAGE.
Su M.Y., Som N., Wu C.Y., Su S.C., Kuo Y.T., Ke L.C., Ho M.R., Tzeng S.R., Teng C.H., Mengin-Lecreulx D., Reddy M, & Chang C.I. (2017). Structural basis of adaptor-mediated protein degradation by the tail-specific PDZ-protease Prc. Nature Communications, 8, 1516.
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5

Recombinant Expression and Purification of SznF

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The sznF gene was amplified from the cosmid encoding the szn gene cluster using the primers in Supplementary Table 4. The amplified gene was digested with NdeI and HindIII and ligated into the pET28a vector as described above for pET28a-SznE. The plasmid was transformed into E. coli BL21 (DE3) cells (Invitrogen), and the protein was expressed and purified using the same procedure as described for SznE with the exception that the exchange buffer contained 20 mM MOPS pH 7.5 instead of 20 mM HEPES pH 8.
Ng T.L., Rohac R., Mitchell A., Boal A.K, & Balskus E.P. (2019). An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin. Nature, 566(7742), 94-99.
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6

Purification and Characterization of NDM and VIM Variants

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The β-lactamases of NDM-1, NDM-5 and VIM-1 were expressed and purified as described previously [20 (link)]. Briefly, the DNA sequences of NDM-1 and NDM-5 were obtained from genomic DNA of the NDM-1-positive isolates E. coli ZC-YN3 and E. coli ZC-YN5, respectively. VIM-1 was synthesized according to the sequence reported on NCBI. The primers used in this study are shown in Table 1. All the DNA sequences were digested by the endonucleases BamHI and XhoI and then cloned into the expression vector pET28a to generate the expression constructs. The gene expression constructs were transferred into E. coli BL21(DE3) cells (Invitrogen). Following induction by Isopropyl-β-D-thiogalactopyranoside (IPTG) for the E. coli BL21(DE3) cells as described above, the water-soluble His-tagged protein was purified from the bacterial lysate by affinity chromatography according to the manufacturer’s instructions. After washing the unbound contaminating proteins with PBS containing 10 mM imidazole, the His-tagged protein was eluted with elution buffer (200 mM imidazole). The protein was concentrated using a filter at 4 °C for desalting, and finally, the purity of the protein was analyzed by SDS-PAGE (DetaiBio, Nanjing, China).
Wang Y., Sun X., Kong F., Xia L., Deng X., Wang D, & Wang J. (2020). Specific NDM-1 Inhibitor of Isoliquiritin Enhances the Activity of Meropenem against NDM-1-positive Enterobacteriaceae in vitro. International Journal of Environmental Research and Public Health, 17(6), 2162.
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7

Recombinant Protein Purification Protocol

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Recombinant constructs were expressed in E.coli BL21 DE3 cells (Invitrogen) in Luria Bertani media. Proteins were purified by affinity chromatography (HisTrap FF, GE Healthcare). The purest fractions were desalted through a PD10 column (GE Healthcare) to remove imidazole before treatment with TEV protease for 4 h at 25°C. The samples were then passed through a second HisTrap column. The cleaved protein was further purified through a Superdex 200 16/600 column (GE Healthcare).
dos Santos Á., Fili N., Hari-Gupta Y., Gough R.E., Wang L., Martin-Fernandez M., Aaron J., Wait E., Chew T.L, & Toseland C.P. (2022). Binding partners regulate unfolding of myosin VI to activate the molecular motor. Biochemical Journal, 479(13), 1409-1428.
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8

Purification of PDE4D5 Mutants

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Full-length human PDE4D5 active mutant S126D and mutant S126D-F268A were subcloned into expression vector pGEX-6P-1 and expressed in E. coli BL21(DE3) cells (Invitrogen). Cells were cultured at 37°C in LB medium until optical density at 600 nm (OD600) reached 0.6. Then, 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) was added to induce protein expression at 30°C for 4 hours. The PDE4D5 mutant proteins were purified using glutathione S-transferase bind resin (Millipore Sigma), glutathione elution, precision protease cleavage, and Superdex 200 (Millipore Sigma) columns. The purified proteins were dissolved in 50 mM tris-HCl buffer (pH 8.0) with 100 mM NaCl and 1 mM β-mercaptoethanol and stored at −80°C. The catalytic domain of human PDE4D5 (317–676) was subcloned into the expression vector pET15b and expressed in E. coli BL21(DE3) cells. Cells were cultured at 37°C until OD600 reached 0.6, when 0.1 mM IPTG was added to induce protein expression at 15°C for 24 hours. The recombinant PDE4D5 catalytic domain was purified using Ni-NTA (nitrilotriacetic acid) affinity columns (Qiagen), thrombin cleavage, and Superdex 200 columns. PDE4D5 catalytic domain protein purity was greater than 95% as shown by SDS–polyacrylamide gel electrophoresis.
Wang S., Xie Y., Huo Y.W., Li Y., Abel P.W., Jiang H., Zou X., Jiao H.Z., Kuang X., Wolff D.W., Huang Y.G., Casale T.B., Panettieri RA J.r., Wei T., Cao Z, & Tu Y. (2020). Airway relaxation mechanisms and structural basis of osthole for improving lung function in asthma. Science signaling, 13(659), eaax0273.
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9

Purification and Co-Immunoprecipitation of Calcineurin

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N-terminally His6-tagged human calcineurin A (α isoform, truncated at residue 400), was expressed in tandem with the calcineurin B subunit in E.coli BL21 (DE3) cells (Invitrogen) and affinity purified using Ni-NTA-agarose with methods identical to the purification of His-tagged yeast calcineurin34 (link). A 16-amino-acid peptide corresponding to HoxB13 residues 255–270, or a mutant version with residues 260, 262, 264 and 265 changed to alanine, or a positive control (Saccharomyces cerevisiae Dig2 residues 95–116 containing a PxIxIT site) were fused to GST in vector pGX4T-3. Cell lysates from bacteria expressing the were obtained as in a previous study34 (link). Bacterial cell lysate (50–100 μg) was used to test co-purification with 2μg of purified His6-calcineurin as described34 (link). Co-purifying proteins were observed by western blotting with mouse GST (Bio Legend) and anti-His monoclonal primary antibodies and an IRDye680RD goat anti-mouse secondary antibody (LiCor) and imaged with the Li-Cor Odyssey imaging system.
Nguyen N.U., Canseco D., Xiao F., Nakada Y., Li S., Lam N., Muralidhar S.A., Savla J., Hill J.A., Le V., Zidan K.A., El-Feky H.W., Wang Z., Ahmed M.S., Hubbi M., Menendez-Montes I., Moon J., Ali S.R., Le V., Villalobos E., Mohamed M.S., Elhelaly W.M., Thet S., Anene-Nzelu C.G., Tan W.L., Foo R., Meng X., Kanchwala M., Xing C., Roy J., Cyert M.S., Rothermel B.A, & Sadek H.A. (2020). A Calcineurin-Hoxb13 Axis Regulates Growth Mode of Mammalian Cardiomyocytes. Nature, 582(7811), 271-276.
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10

Purification of Recombinant Human DAAO

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The cDNA coding for hDAAO was cloned in the pET11b expression plasmid, under control of the T7 promoter. The His-tagged recombinant protein was expressed in E. coli BL21 (DE3) cells (Invitrogen, Carlsbad, CA, United States) upon induction with IPTG during the exponential phase of growth and purified by HiTrap chelating chromatography (GE Healthcare, Boston, MA, United States), as reported in (Murtas et al., 2017 (link)). For long storage at −80°C, the final protein preparation was equilibrated in 20 mM Tris–HCl buffer, 100 mM NaCl, pH 8.0, 10% glycerol, and 5 mM 2-mercaptoethanol, to which 40 μM FAD was added. The concentration of the purified enzyme was determined using the extinction coefficient at 455 nm (12.2 mM−1 cm−1) (Molla et al., 2006 (link)).
Sacchi S., Rabattoni V., Miceli M, & Pollegioni L. (2021). Yin and Yang in Post-Translational Modifications of Human D-Amino Acid Oxidase. Frontiers in Molecular Biosciences, 8, 684934.
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