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Bl21 de3

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BL21 (DE3) is a bacterial strain commonly used in molecular biology and protein expression experiments. It is an E. coli strain that has been genetically modified to enhance the expression of recombinant proteins. The strain harbors the DE3 lysogen, which contains the T7 RNA polymerase gene under the control of the lacUV5 promoter, allowing for inducible protein expression.

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Lab products found in correlation

Biotinylation kit, by Thermo Fisher Scientific (1 mentions) Hitrap hp column, by GE Healthcare (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Pd 10 column, by GE Healthcare (1 mentions) Pcr2.1 plasmid, by Thermo Fisher Scientific (1 mentions) Pqe30 plasmid, by Qiagen (1 mentions)

3 protocols using bl21 de3

1

Production and Purification of PspA and PspA-C-CPE Proteins

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pET16b plasmids encoding PspA or PspA-C-CPE were prepared as previously described11 (link). To obtain recombinant protein, plasmids were transformed into Escherichia coli strain BL21 (DE3) (TOYOBO, Osaka, Japan). To induce the production of PspA or PspA-C-CPE, isopropyl-D-thiogalactopyranoside (Nacalai Tesque, Kyoto, Japan) was added to the culture medium. The culture pellet was sonicated in buffer A (10 mM Tris–HCl [pH 8.0], 400 mM NaCl2, 5 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol, and 10% glycerol). The supernatant was loaded onto a HiTrap HP column (GE Healthcare, Pittsburgh, Pennsylvania, USA). PspA or PspA-C-CPE protein was eluted with buffer A containing 100 to 500 mM imidazole. The solvent was exchanged with phosphate-buffered saline (PBS) by using a PD-10 column (GE Healthcare). The concentration of recombinant protein was measured by using a BCA Protein Assay Kit (Life Technologies, Carlsbad, California, USA). PspA-C-CPE was biotinylated by using a biotinylation kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Suzuki H., Nagatake T., Nasu A., Lan H., Ikegami K., Setou M., Hamazaki Y., Kiyono H., Yagi K., Kondoh M, & Kunisawa J. (2018). Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a claudin-4-targeting nasal vaccine in mice. Scientific Reports, 8, 2904.
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2

Recombinant Protein Expression of PxXyl43A

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P. xylaniclasticus strain TW1 was isolated previously from the wastes of a pineapple processing factory in Thailand.5) (link) The Escherichia coli strains αINV (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and ME9806 (iVEC3) (National BioResource Project (NBRP), Mishima, Japan) were used as cloning hosts while E. coli JM109 and BL21(DE3)(TOYOBO CO., LTD., Japan) were used as protein expression hosts.7) (link) The pCR2.1 plasmid (Invitrogen) was used for cloning, and the pQE30 plasmid (QIAGEN Benelux B.V., Venlo, Netherland) and pET16b plasmid (Novagen Inc., Madison, WI, USA) were used for expression of recombinant His-tagged proteins. Transformed E. coli was cultivated in LB liguid medium supplemented with ampicillin (50 µg/mL). The recombinant proteins of the full length of PxXyl43A (PxXyl43A) and the unknown function module at the C-terminus of PxXyl43A (PxXyl43A-UM) were expressed using the plasmids pET16b-PxXyl43A and pQE30-PxXyl43A-UM, respectively.
Ito D., Nakano E., Karita S., Umekawa M., Ratanakhanokchai K, & Tachaapaikoon C. (2022). Characterization of a GH Family 43 β-Xylosidase Having a Novel Carbohydrate-binding Module from Paenibacillus xylaniclasticus Strain TW1. Journal of Applied Glycoscience, 69(3), 65-71.
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3

Anaerobic Biofuel Cultivation Protocols

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Clostridium thermocellum DSM 1313 was anaerobically cultured in CTFUD medium with cellobiose or cellulose at 55 • C (Olson and Lynd 2012b). Transformed C. thermocellum cells were cultured in CTFUD medium supplemented with 24 μg mL -1 of thiamphenicol. Escherichia coli JM109 and BL21(DE3) (TOYOBO) were cultured in Luria broth supplemented with 50 μg mL -1 of ampicillin or 20 μg mL -1 of chloramphenicol at 37 • C.
Ichikawa S, & Karita S. (2015). Bacterial production and secretion of water-insoluble fuel compounds from cellulose without the supplementation of cellulases. FEMS microbiology letters, 362(24).
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