Construction of Plasmids for Innolysin Cj Expression

Coding sequences for Innolysins Cj1 and Innolysin Cj2 were chemically synthesized by GeneCust (Luxembourg) and cloned into pET-28a (+) using the NdeI and XhoI restriction sites (Table 3). Plasmid pAZInCj1 expressing Innolysin Cj1 was designed by fusing the coding sequence of H-fiber protein of CJIE1-like prophage originating from CAMSA2147 ({"type":"entrez-protein","attrs":{"text":"WP_002878910.1","term_id":"488967918","term_text":"WP_002878910.1"}}WP_002878910.1) to the C-terminus of phage T5 endolysin coding sequence ({"type":"entrez-protein","attrs":{"text":"YP_006868.1","term_id":"46401777","term_text":"YP_006868.1"}}YP_006868.1). A linker encoding 14 amino acids (Linker 2, Supplementary Table S2) was used to join both coding sequences. The same configuration was used for plasmid pAZInCj2 expressing Innolysin Cj2, but instead of the whole H-fiber, the C-terminal part of H-fiber coding sequence was used, excluding the N-terminal DUF3751 domain. Both plasmids pAZInCj1 and pAZInCj2 also contain the flanking downstream gene of CAMSA2147, the putative fiber chaperone ({"type":"entrez-protein","attrs":{"text":"WP_002878909.1","term_id":"488967917","term_text":"WP_002878909.1"}}WP_002878909.1). In-Fusion^® cloning was used to clone either the T5 endolysin or the C-terminus (amino acids 151 to 343) of the H-fiber ({"type":"entrez-protein","attrs":{"text":"WP_002878910.1","term_id":"488967918","term_text":"WP_002878910.1"}}WP_002878910.1) along with the downstream chaperone gene ({"type":"entrez-protein","attrs":{"text":"WP_002878909.1","term_id":"488967917","term_text":"WP_002878909.1"}}WP_002878909.1) into peT28a (+), giving rise to the control plasmids pAZE1 or pCRYS3, respectively (Table 3). Amplification of these genes was performed with specific primers (Supplementary Table S1) and using phage T5 (Leibniz Institute DSMZ) or C. jejuni CAMSA2147 DNA as a template, respectively. The vector pET28a (+) was linearized using NdeI and XhoI restriction enzymes and insertion was performed based on the In-Fusion^® HD cloning manual. Plasmids were obtained after transformation of E. coli Stellar competent cells and selection on LB agar with kanamycin (100 μg/ml). Subsequently, all plasmids were used to transform E. coli BL21-CodonPlus(DE3)-RIL (Agilent Technologies) by plating on LB agar plates in the presence of kanamycin (100 μg/ml) and chloramphenicol (50 μg/ml). All constructs were sequenced by Sanger sequencing.