Ndei and xhoi

Primers with overhanging NdeI and XhoI sequences (Table S5) were designed to PCR amplify the nucleotide sequence of effector genes of interest from the pJWC1 insert template cosmid with Q5 High-Fidelity DNA polymerase (NEB) according to the manufacturer’s protocol. The annealing temperature was calculated for each primer pair using the online NEB Tm Calculator (version 1.9.13), and extension time was set according to the expected amplicon length (30 s/kb). PCR products were gel purified and digested with NdeI and XhoI (NEB), ligated into NdeI- and XhoI-digested and dephosphorylated pET28c vector using T4 DNA ligase (NEB), and transformed into electrocompetent T7 express E. coli. Single colonies were cultured in LB medium with 50 μg/ml kanamycin, and plasmid DNA was extracted using a Monarch Plasmid Miniprep kit (NEB). Identity of the cosmid inserts was confirmed by Sanger sequencing using T7 and T7-term specific primers (Table S5). The subclones were cultured in LB medium in the presence of kanamycin (50 μg/ml) and, upon reaching an optical density at 580 nm (OD₅₈₀) of 0.6, induced with isopropyl-β-D-thiogalactopyranoside (IPTG; 500 μM) for 20 h at 18°C. Sterile spent broth collected after this period was tested for activity using the NF-κB assay as described above.

The E. coli agmatinase amino acid sequence (UniProt P60651) was first codon optimized for expression in an E. coli expression system using the GenSmart codon optimization tool (GenScript) (S1 Fig in S1 File). The gene was synthesized (GenScript) with a 5′ NdeI restriction endonuclease site and a stop codon followed by a XhoI restriction endonuclease site at the 3′ terminus, and delivered in a pUC57 vector (SPEB-pUC57). The gene was subcloned into the pTHT vector, a variant of pET-28 with a tobacco etch virus (TEV) protease site in place of the thrombin site. Briefly, SPEB-pUC57 and pTHT were separately digested with NdeI and XhoI (NEB). The digested gene and plasmid were separated on an agarose gel, gel purified, and then ligated using T4 DNA ligase (NEB). The ligated product was used to transform 5-alpha E. coli cells (NEB) and plated on LB agar supplemented with 50 μg/mL kanamycin. Selected colonies were cultured and the resulting plasmids (SPEB-THT) isolated for sequence verification by Sanger sequencing.