Bl21 gold de3

Manufactured by Thermo Fisher Scientific

BL21-Gold (DE3) is a competent Escherichia coli strain commonly used for the expression of recombinant proteins. It is designed to enhance the production of heterologous proteins through optimized expression conditions.

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Lab products found in correlation

Xl10 gold, by Thermo Fisher Scientific (2 mentions) T4 dna ligase, by Thermo Fisher Scientific (2 mentions) Restriction enzyme, by New England Biolabs (2 mentions) Fluo 3, by Thermo Fisher Scientific (1 mentions) Gcamp6f, by Addgene (1 mentions) Ez run protein ladder, by Thermo Fisher Scientific (1 mentions) Human plasma thrombin, by Merck Group (1 mentions)

Close spelling variants of this product

BL21 (DE3) Gold cells BL21(DE3)

5 protocols using bl21 gold de3

Plasmid-based Fluorescent Biosensors

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GCaMP6f, G-GECO 1.0 and GEM-GECO plasmids were purchased from Addgene (plasmid #40755, #32466 and #32463, respectively). pET41a and pEGFP-N1 vectors were obtained from Novagen and Clontech, respectively. XL10-Gold and BL21 (DE3) Gold cells were purchased from Invitrogen. Fluo-3 was purchased from Molecular Probes and EZ-Run protein ladder from Fisher Bioreagents. Restriction enzymes were obtained from New England Biolabs and T4 DNA ligase from Fermentas. Br₂-BAPTA was purchased from Life Technologies.

Helassa N., Zhang X.H., Conte I., Scaringi J., Esposito E., Bradley J., Carter T., Ogden D., Morad M, & Török K. (2015). Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics. Scientific Reports, 5, 15978.

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Plasmid Cloning and Cell Lines

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GCaMP6s and GCaMP6f plasmids were a gift from Douglas Kim (Addgene plasmid #40753 and #40755, respectively)19 (link). pET41a and pEGFP-N1 vectors were obtained from Novagen and Clontech, respectively. XL10-Gold and BL21 (DE3) Gold cells were purchased from Invitrogen. Restriction enzymes were obtained from New England Biolabs and T4 DNA ligase from Fermentas.

Helassa N., Podor B., Fine A, & Török K. (2016). Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. Scientific Reports, 6, 38276.

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Bacterial Strains for Subcloning, Phage Library, and Protein Purification

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Escherichia coli (E.coli) XL1-Blue or DH5α were used for subcloning and plasmid amplification, which were grown in LB medium at 37°C. E.coli TG1 (Lucigen, 60502) was used for phage library creation and phage propagation. TG1 cells were grown in 2x YT medium at 37°C to reach an OD₆₀₀ of 0.4, followed by addition of eluted phage (50 μL per 25 mL culture). E.coli BL21 Gold DE3 (Invitrogen, C600003) or Rosetta DE3 (Novagen, 70954) were used for protein purification. Generally, cells were grown at 37°C until OD₆₀₀ of 0.6 to 0.8, followed by induction with 0.4–1 mM isopropyl β-d-thiogalactoside (IPTG) at 18 or 20°C for 16–20 h.

Luo Y., Schofield J.A., Na Z., Hann T., Simon M.D, & Slavoff S.A. (2020). Discovery of cellular substrates of human RNA-decapping enzyme DCP2 using a stapled bicyclic peptide inhibitor. Cell chemical biology.

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Isotopically Labelled Protein Expression

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¹H, ¹⁵N, ¹³C, labelled mt AcP and Sso AcP were expressed as a GST fusion proteins in the E. coli strain BL21-Gold (DE3) (Invitrogen), grown in minimal medium by using ¹⁵N- ammonium chloride and ¹³C-glucose. Purification of mAcP and SsoAcP were then performed following previous protocols (Taddei et al., 1996 (link)). Cell lysates were purified using a glutathione column (Sigma Aldrich) following GST cleavage with human plasma thrombin (Sigma Aldrich) in TRIS buffer. Eluted proteins were then buffer exchanged into 30 mM ammonium carbonate buffer at pH 8.0 and then lyophilized.

Fusco G., Bemporad F., Chiti F., Dobson C.M, & De Simone A. (2022). The role of structural dynamics in the thermal adaptation of hyperthermophilic enzymes. Frontiers in Molecular Biosciences, 9, 981312.

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Overexpression of E. coli 30S proteins

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The genes for the E. coli 30S 3’domain r-proteins were cloned into a pRSF-1b expression vector (Novagen) and overexpressed in BL21-Gold (DE3) (Invitrogen) or Tuner™ (DE3) E. coli strains (Novagen). E. coli strains were grown in LB medium at 37 °C in culture flasks ranging in volume from 5 ml test tubes to 2 L baffled flasks.

Duss O., Stepanyuk G.A., Puglisi J.D, & Williamson J.R. (2019). Transient protein-RNA interactions guide nascent ribosomal RNA folding. Cell, 179(6), 1357-1369.e16.

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