One shot bl21 de3 plyss chemically competent e coli cells

The entire bosR-coding region was amplified from genomic DNA of B. burgdorferi B31 with the use of primers P5F and P5R (Table 1). The resultant PCR product was digested, purified and cloned into pET-23a vector (EMD Chemicals Inc., Darmstadt, Germany), generating a construct that contained the bosR-coding region fused with a C-terminal His₆ tag. One Shot BL21(DE3)pLysS Chemically Competent E. coli cells (Life Technologies, Grand Island, NY) were transformed with the construct and induced with 1.0 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) (Sigma Chemical Co., St. Louis, MO). Recombinant BosR was affinity-purified with the use of HiTrap Chelating HP following the manufacturer’s instruction (GE Healthcare Bio-Sciences, Pittsburgh, PA). Protein concentration was measured using Quick Start Bradford Dye Reagent following the manufacturer’s protocol (Bio-Rad Laboratories, Hercules, CA).

The plasmid pPlySK1249^28a (25 (link)) was used as a template for PCR amplification of genes encoding for truncated forms of the PlySK1249 endolysin (Fig. 1A) using specific primer pairs listed (Microsynth AG). PCR products were digested using restriction enzymes NcoI and XhoI (Promega) and ligated into predigested expression vector pET28a. Obtained plasmids (namely pAmi^28a, pAmi_LysM^28a, and pLysM_CHAP^28a, Table S1) were transformed in One Shot BL21 (DE3) pLysS chemically competent E. coli cells (Life Technologies Europe B.V.). Plasmid DNA was purified using the Qiagen Miniprep kit following the manufacturer’s recommendations and all constructs were validated by DNA sequencing using universal T7 primers (Table S1). Following 0.4 mM IPTG induction for 18 h, recombinant proteins were purified by affinity chromatography as described previously (25 (link)) and loaded on NuPAGE 4 to 12% BisTris gels (Invitrogen) to confirm the correct molecular weights and assess their purity.