Bl21star de3 prare

The purified plasmids containing the bla_OXA-48 gene were transformed to E. coli strain BL21Star(DE3)pRARE [Invitrogen, Novagen and [25 (link)] competent cells by a conventional heat-shock protocol. Cells were used to inoculate 1 L Terrific Broth (TB) media containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Recombinant expression of native OXA-48 was induced by 0.4 mM isopropyl-β-D-thiogalactopyranoside (VWR) at log phase, and expression was continued at 20°C for 16 hours. OXA-48 was isolated from the periplasm using osmotic shock and lysozyme-treatment [26 (link)], and purified through two anionic exchange steps, as described previously [27 ].

The Escherichia coli strains used in this study were DH5α and XL10-gold for plasmid propagation and BL21 star (DE3) pRARE (Invitrogen, Carlsbad, CA) for protein expression. C. thermophilum was obtained from NBRC (Biological Resource Center, National Institute of Technology and Evaluation, Tokyo, Japan). The concentrations of CtCCTs were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard, and the concentrations are reported as molar concentrations of hexadecamers. The NucleoSpin RNA Plant (Takara Bio Inc., Shiga, Japan) and cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan) were used for the RNA and cDNA work. KOD-Plus-Neo DNA polymerase was used for gene amplification, and restriction endonucleases were obtained from Toyobo (Osaka, Japan) and New England Biolabs Japan (Tokyo, Japan). The site-directed mutagenesis of CtCCTs was performed using the QuikChange site-directed mutagenesis kit and QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Nucleotides and other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) or Roche and Sigma-Aldrich Japan.