E coli bl21 de3 plyss competent cells

Manufactured by Thermo Fisher Scientific

E. coli BL21(DE3) pLysS competent cells are a strain of Escherichia coli bacteria that have been genetically modified to facilitate the expression of recombinant proteins. These cells contain the DE3 lysogen, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter, and the pLysS plasmid, which expresses T7 lysozyme to reduce basal expression of the target protein.

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Lab products found in correlation

Pet28a expression vector, by Thermo Fisher Scientific (1 mentions) Coomassie brilliant blue r 250, by Merck Group (1 mentions)

Close spelling variants of this product

BL21(DE3) (201 citations) E. coli BL21 (DE3) (131 citations) E. coli BL21 (DE3) cells (74 citations) BL21(DE3)pLysS (66 citations) BL21(DE3) cells (59 citations) E. coli BL21 (26 citations) BL21(DE3)pLysS cells (20 citations) E. coli BL21 (DE3) pLysS (18 citations) BL21 (DE3) competent cells (16 citations) BL21 (DE3) pLysS E. coli cells (13 citations)

2 protocols using e coli bl21 de3 plyss competent cells

Protein Analysis by SDS-PAGE

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Protein analysis by SDS-PAGE was performed using 15% and 5% acrylamide in the resolving and stacking gels, respectively. Gels were stained with Coomassie brilliant blue R-250 (Sigma-Aldrich). Electrophoresis was run under reducing conditions in the presence of 5% β-mercaptoethanol. Pyruvate kinase (PK), lactate dehydrogenase (LDH), ATP, and 2-keto-D-gluconate (KG) were purchased from Sigma-Aldrich. 2-keto-3-deoxygluconate (KDG) was prepared as described by Lamble et al. [38 (link)] using a pyruvate aldolase from Sulfolobus solfataricus. 2-ketogulonate (KGul), 2-keto-3-deoxygulonate (KDGul), and 2-keto-d-galactonate (KGal) were prepared as described by de Berardinis et al. [37 (link)]. E. coli BL21(DE3) pLysS competent cells and pET28a expression vector were purchased from Invitrogen. Plasmid DNA purification kits were from Sigma-Aldrich. All other chemicals were purchased from Sigma-Aldrich as reagent grade.

Sánchez-Moreno I., Trachtmann N., Ilhan S., Hélaine V., Lemaire M., Guérard-Hélaine C, & Sprenger G.A. (2019). 2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity. Molecules, 24(13), 2393.

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Overexpression and Purification of V6

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The plasmid for encoding V6 was transformed into E. coli BL21-(DE3)-pLysS competent cells (Invitrogen, Carlsbad, CA) by heat shock at 42 °C. The transformed cells were then incubated and grown at 37 °C to log phase when the optical density at 600 nm reaches 1.0, after that, Isopropyl-β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 1 mM, was added to the cell culture to induce the protein expression. Expression time was optimized by the pilot expression. The V6 construct was purified from the inclusion body as described previously^{15 , 77 (link)} with one modification considering the different isoelectric point (pI) for V6 of 8.92.

Liu Z., Casey T.M., Blackburn M.E., Huang X., Pham L., de Vera I.M., Carter J.D., Kear-Scott J.L., Veloro A.M., Galiano L, & Fanucci G.E. (2016). Pulsed EPR Characterization of HIV-1 Protease Conformational Sampling and Inhibitor-Induced Population Shifts. Physical chemistry chemical physics : PCCP, 18(8), 5819-5831.

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