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E coli bl21 cells

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E. coli BL21 cells are a widely used bacterial expression strain. They are commonly employed for the production of recombinant proteins in laboratory settings.

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Lab products found in correlation

Ptrchis topo, by Thermo Fisher Scientific (1 mentions) Ptrchis, by New England Biolabs (1 mentions) Phenylmethylsulfonyl fluoride pmsf, by Roche (1 mentions) Kanamycin, by Merck Group (1 mentions) Hepes free acid, by Research Products International (1 mentions) Gmppnp, by Abcam (1 mentions) Bacto yeast extract, by Thermo Fisher Scientific (1 mentions) L glutathione reduced, by Merck Group (1 mentions) Ampicillin, by Research Products International (1 mentions) Sds page, by Bio-Rad (1 mentions) Illustrator cc 2015, by Adobe (1 mentions) Bio safe coomassie blue, by Bio-Rad (1 mentions) Recombinant kinase, by PerkinElmer (1 mentions) Glutathione sepharose 4b, by Thermo Fisher Scientific (1 mentions) 32p atp, by PerkinElmer (1 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)

6 protocols using e coli bl21 cells

1

Recombinant Expression of Bacterial Flagellins

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Synthetic genes encoding A-type (GenBank accession no. CP020659.1) flagellin from PAK and B-type (GenBank accession no. AE004091.2) flagellin from PAO1 were codon optimized by GenScript (NJ) for expression in E. coli (S2 Table). Both synthetic genes were cloned into pTrc-His-TOPO (Thermo Fisher, MA) after PCR amplification using primers fla_F and fla_R (S1 Table). The resulting plasmids, pTrcHis-flaAopt and pTrcHis-flaBopt, were then transformed into E. coli BL21 cells (New England Biolabs, MA) according to the manufacturers protocol, for production of A-type and B-type flagellin, respectively, as recombinant proteins incorporating a 6xHis tag.
Hegerle N., Choi M., Sinclair J., Amin M.N., Ollivault-Shiflett M., Curtis B., Laufer R.S., Shridhar S., Brammer J., Toapanta F.R., Holder I.A., Pasetti M.F., Lees A., Tennant S.M., Cross A.S, & Simon R. (2018). Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa. PLoS ONE, 13(9), e0203143.
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2

Ras Protein Purification from E. coli

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E. coli BL21 cells were obtained from New England Biolabs (Ipswich, MA), and Bacto yeast extract, agar and tryptone for bacterial growth were from Gibco (Waltham, MA). Ampicillin and HEPES free acid were from Research Products International (Mount Prospect, IL). Kanamycin and reduced L-glutathione were from Sigma-Aldrich (St. Louis, MO). Phenylmethylsulfonyl fluoride (PMSF) used to inhibit proteolysis during Ras purification was from Roche Diagnostics (Basel, Switzerland). TALON cobalt(II) metal affinity resin for polyhistidine tag purification was from Takara (Kusatsu Japan), and HYDRANAL imidazole was from Honeywell (Charlotte, NC). Ethylenediamine tetracetic acid disodium salt dihydrate (EDTA) was from Thermo Fisher Scientific (Waltham, MA). Non-hydrolyzable GTP analog GMPPNP, conjugated with four lithium counter ions, at >95% purity, was from Abcam (Cambridge, UK). Guanosine 5’-diphosphate [GDP] disodium salt was from Sigma-Aldrich (St. Louis, MO). Desalting columns were Econo-Pac 10DG desalting prepacked gravity flow columns from Bio-Rad (Hercules, CA). Vivaspin 500, 10,000 MWCO spin concentrators from Sartorius were used to concentrate protein samples (Göttingen, Germany). Microscale thermophoresis measurements were performed on a Nanotemper Monolith NT.115 instrument utilizing Monolith NT.115 capillaries (Munich, Germany).
Fleming I., Hannan J.P., Swisher G.(., Tesdahl C.D., Martyr J.G., Cordaro N.J., Erbse A.H, & Falke J.J. (2022). Binding of Active Ras and Its Mutants to the Ras Binding Domain of PI-3-Kinase: A Quantitative Approach to KD Measurements. Analytical biochemistry, 663, 115019.
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3

Recombinant VHH Expression and Purification

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The selected VHHs expressed in pHEN1 plasmid were transformed into E. coli BL21 cells (NEB, Ipswich, MA, USA) for expression and purification. An overnight culture was obtained at 37 °C, in a culture shaker at 210 rpm. The next day, the cells were harvested by centrifugation, lysed by BugBuster Protein Extraction Reagent (Novagen, Madison, WI, USA), and VHHs were purified from the lysate by TALON Superflow containing Co2+ agarose (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The eluted fraction was subjected to dialysis with Visking dialysis tubing (MWCO 12000–14000) (Serva, Heidelberg, Germany). VHHs were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA) under denaturing conditions and had an expected molecular weight of ~15 kDa.
Godakova S.A., Noskov A.N., Vinogradova I.D., Ugriumova G.A., Solovyev A.I., Esmagambetov I.B., Tukhvatulin A.I., Logunov D.Y., Naroditsky B.S., Shcheblyakov D.V, & Gintsburg A.L. (2019). Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice. Toxins, 11(8), 464.
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4

Bacterial Induction Imaging Protocol

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All agar plate inductions were carried out on LB agar (Miller) supplemented with 100 μg/mL ampicillin. All cells were transformed into BL21 E. coli cells (New England Biolabs), plated on LB/agar plates, and grown overnight at 37°C to produce single colonies. Colonies from these plates were picked and streaked generously to fill in guided lines on fresh LB/agar plates using a pipette tip that was sterilely bent 90° for streaking bacteria. Templates were printed and taped to the bottom of agar plates to help ensure consistency (0.75 cm radius circle with 3 cm long × 1 cm wide curved rectangles 0.2 cm from circle equally spaced around the circle). Plates were imaged on a Pxi4 imager at their respective time points using the 488 nm blot setting with automatic image focus and exposure. 2D images were converted to 3D using the Pxi4 software. Images were saved as TIFFs and induction distances were analyzed on Adobe Illustrator CC 2015.
Tekel S.J., Smith C.L., Lopez B., Mani A., Connot C., Livingstone X, & Haynes K.A. (2019). Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli. Frontiers in Bioengineering and Biotechnology, 7, 80.
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5

Cpx7A Mutagenesis and Kinase Assay

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QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) was used for site-directed mutagenesis of unedited Cpx7A to generate Cpx7AI125M,N130S (termed N130S). Cpx variants were subcloned into a pGEX-2T construct (GE Healthcare Life Sciences) and the recombinant Cpx variants fused with GST were expressed in BL21 E. coli cells (New England BioLabs) and purified using glutathione Sepharose 4B (Thermo Fisher Scientific). Peak fractions were concentrated and further purified by gel filtration as previously described.10 (link)In vitro kinase assays were performed using purified recombinant Cpx proteins and the catalytic subunit of CK2 (C70-10G; SignalChem). Briefly, 10 mg of purified GST-fusion protein (unedited Cpx7AI125,N130 or edited Cpx7AI125M,N130S) was used per reaction and incubated with 2,500 units of recombinant kinase and [32P]ATP (PerkinElmer). Reaction products were separated by SDS-PAGE and gels were stained with Bio-Safe Coomassie Blue (Bio-Rad), dried, and exposed to autoradiography film at room temperature. Mean integrated density of each band was quantified using FIJI and relative density of phospho-Cpx (pCpx) was calculated by normalizing to input band intensity determined by Coomassie staining.
Jalving R., van de Vondervoort P.J., Visser J, & Schaap P.J. (2000). Characterization of the Kexin-Like Maturase of Aspergillus niger. Applied and Environmental Microbiology, 66(1), 363-368.
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6

Purification of GST-Fusion Protein

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Glutathione-S-transferase (GST) fusion protein was purified as follows: Briefly, pGEX-6P1 plasmid containing repeated uba1 domains of human HR23A [65 (link)] was transformed into BL21 E. coli cells (New England Biolabs) and colonies were obtained by selective growth in solid Luria Bertani medium (LB) plus ampicillin at 37°C.
Expression of GST-fusion protein was induced with 0.5 mM IPTG for 2 h at 37°C. The bacteria were centrifuged at 3,000g for 15 min at 4°C and washed in ice-cold PBS. After centrifugation, the pelleted bacteria were lysed during a 30-min incubation in 25 ml ice-cold PBS containing 1 mg/ml lysozyme and 1 mM AEBSF. The lysate was then sonicated, successively syringed though a 18-, 23-, and 25-gauge needles and centrifuged at 10,000g for 30 min to remove debris. The supernatant (that contains soluble GST-TUBE; 800 μl) was 0.45-μm filtered and incubated with equilibrated glutathione-agarose beads (Clinisciences; 70 μl) for 30 min at room temperature, with gentle agitation and the beads were rinsed 3 times with ice-cold PBS.
Chesnel F., Couturier A., Alusse A., Gagné J.P., Poirier G.G., Jean D., Boisvert F.M., Hascoet P., Paillard L., Arlot-Bonnemains Y, & Le Goff X. (2020). The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation. PLoS Genetics, 16(11), e1009183.
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