E coli bl21 de3 competent cells

Manufactured by Agilent Technologies

Sourced in United States

E. coli BL21(DE3) competent cells are a widely used strain of Escherichia coli bacteria that are genetically modified to facilitate the expression of recombinant proteins. These cells are designed to enhance the production of target proteins in a controlled and efficient manner.

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Lab products found in correlation

Oligonucleotide primer, by Merck Group (1 mentions) Restriction enzyme, by New England Biolabs (1 mentions) Gstrap ff column, by Cytiva (1 mentions) Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions) Bradford protein assay, by Bio-Rad (1 mentions) Pd 10 column, by Cytiva (1 mentions) Nucleodur c18 pyramid column, by Macherey-Nagel (1 mentions) Fs hydrodex β 6tbdm, by Macherey-Nagel (1 mentions) Glutathione sepharose 4b resin, by GE Healthcare (1 mentions) Fungizone, by Merck Group (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Penicillin g, by Merck Group (1 mentions) Gentamicin, by Merck Group (1 mentions)

4 protocols using e coli bl21 de3 competent cells

Cloning and Protein Expression Protocols

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Plasmids and E. coli competent cells for cloning were purchased from Novagen and New England BioLabs. The E. coli BL21 (DE3) competent cells used for protein expression were from Agilent. All chromatography columns were from GE Healthcare. Hotstart Pfu polymerase was from Agilent. Restriction enzymes were from New England BioLabs. Oligonucleotide primers were sourced from Sigma. Substrate S1P (CAS number: 26993-30-6) was from Cayman Chemical Co. 2E-HEX was synthesized in house using a published method (29 ). All other buffers and reagents were sourced from Sigma.

McLean C.J., Marles-Wright J., Custodio R., Lowther J., Kennedy A.J., Pollock J., Clarke D.J., Brown A.R, & Campopiano D.J. (2016). Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei. Journal of Lipid Research, 58(1), 137-150.

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Purification of Recombinant Plasmodium Kinases

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The full coding sequencing (CDS) of Pf NEK3, aside from the first 10 residues on the N-terminus, cloned in a pGEX-4T-3 vector, was a generous gift from Christian Doerig. The PfARK2-GST construct was generated within the Chakrabarti laboratory, and cloning is described in the Supporting Information. E. coli BL21(DE3) competent cells (Agilent, Santa Clara, CA) were transformed via heat shock, and expression was induced with 1 mM IPTG at 20 °C for 16 h. Bacterial lysates were pelleted by centrifugation and resuspended in a HEPES salt buffer (50 mM HEPES, 200 mM NaCl, 0.02% monothiolglycerol, pH 7.5) supplemented with 1× HALT Protease Inhibitor (#78443, Thermo Fisher, Waltham, MA). Lysis was achieved with French press and sonication, and the lysate was clarified with centrifugation and filtration through a 0.2 μm PES membrane. Total protein lysate was run through a GSTrap FF Column (Cytiva, Marlborough, MA) and eluted with the suspension buffer supplemented with 20 mM glutathione. The resulting fractions were desalted with PD-10 Columns (Cytiva, Marlborough, MA) and quantified with a Bradford protein assay in accordance with the manufacturer’s protocol (BioRad, Hercules, CA).

Bohmer M.J., Wang J., Istvan E.S., Luth M.R., Collins J.E., Huttlin E.L., Wang L., Mittal N., Hao M., Kwiatkowski N.P., Gygi S.P., Chakrabarti R., Deng X., Goldberg D.E., Winzeler E.A., Gray N.S, & Chakrabarti D. (2023). Human Polo-like Kinase Inhibitors as Antiplasmodials. ACS infectious diseases, 9(4), 1004-1021.

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Enantioselective Enzymatic Synthesis

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Substrates 1 were synthesized as described in the Supporting Information. All other chemicals used in this study were purchased from commercial suppliers. A Bruker (Rheinstetten, Germany) DPX‐200 NMR device was used for ¹H and ¹³C NMR measurements. High performance liquid chromatography (HPLC) analyses were performed with an AZURA HPLC System (Knauer, Berlin, Germany) with a NUCLEODUR C18 Pyramid column (5 μm; 4.6×250 mm; Macherey–Nagel, Düren, Germany). The enantiomeric excess of intermediates 2 and products 3 was determined by chiral gas chromatography with a flame ionization detector (GC‐FID) by using a Shimazu GC Plus 2010 device (Shimazu, Kyoto, Japan) with a chiral column FS‐Hydrodex‐β‐6TBDM (Macherey–Nagel, Düren, Germany).
The codon‐optimized genes of B. bronchiseptica AMD‐WT (UniProtKB/Swiss‐Prot: Q05 115, PDB: 3DG9) and its variants V43I/A125P/V156L/M159L (AMD‐IPLL), G74C/M159L/C188G (AMD‐CLG), and V43I/G74C/A125P/V156L/M159L/C188G (AMD‐CLGIPL) were cloned into a pET28a vector with treatment of restriction enzymes NdeI, and XhoI. E. coli BL21(DE3) competent cells (Agilent Technologies, Santa Clara, CA, USA) were used for protein expression.22

Enoki J., Mügge C., Tischler D., Miyamoto K, & Kourist R. (2019). Chemoenzymatic Cascade Synthesis of Optically Pure Alkanoic Acids by Using Engineered Arylmalonate Decarboxylase Variants. Chemistry (Weinheim an Der Bergstrasse, Germany), 25(19), 5071-5076.

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Osteoclastogenesis in RAW264.7 Cells

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OCG in RAW264.7 cells (passages 5–15; American Type Culture Collection, Manassas, VA, USA) was initiated by plating 5×10⁴ cells into each well of an 8-chamber BD Falcon glass culture slide (BD Biosciences Discovery Labware, Bedford, MA, USA). The cells were incubated in 0.7 ml Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 10% antibiotic/antimycotic solution (164 IU/ml penicillin G, 50 μg/ml gentamicin, and 0.25 μg/ml fungizone; Sigma-Aldrich, Oakville, ON, Canada). Once cells were plated in the dishes, sRANKL was added at a final concentration of 60 ng/ml. sRANKL was purified from E. coli BL21(DE3) competent cells (Agilent Technologies, La Jolla, CA, USA) transformed with pGEX-4T-1-sRANKL (kindly provided by Dr M.F. Manolson) using glutathione sepharose 4B resin (GE Healthcare, Piscataway, NJ, USA). All the incubations were performed at 37°C in 5% CO₂ humidified air for 4 days. Culture media was changed and sRANKL was supplemented on the second day in culture.

QI W., GAO Y., TIAN J, & JIANG H. (2014). Adseverin knockdown inhibits osteoclastogenesis in RAW264.7 cells. International Journal of Molecular Medicine, 34(6), 1483-1491.

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