Bl21 de3

E. coli strain BL21 (DE3) (Nippon Gene) was transformed with pET21c-mazF_NE1181 via heat shock, and this transformant was pre-cultivated overnight in LB medium supplemented with 100 μg/mL ampicillin at 37 °C. Pre-cultivated E. coli cells were then inoculated into 1 L LB medium containing 100 μg/mL ampicillin and 3% NaCl and then incubated overnight. MazF_NE1181 was induced by the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside. After 3.5 h of incubation, the cells were harvested by centrifugation at 7000 g and then stored at −80 °C until use.

pET19b‐mazE_DR0416 was introduced into the E. coli strain BL21 (DE3) (BioDynamics Laboratory Inc., Tokyo, Japan), whereas pET21c‐mazF_DR0417 was introduced into the strain BL21 (DE3) (Nippon Gene) using the heat‐shock method. The E. coli cells harboring pET19b‐mazE_DR0416 or pET21c‐mazF_DR0417 were grown overnight in liquid LB medium supplemented with 100 μg/mL ampicillin at 37°C. These cells were inoculated into 1 L LB medium containing 100 μg/mL ampicillin. One millimolar IPTG was added to induce MazE_DR0416 and MazF_DR0417 when the OD₆₀₀ reached 0.8 and 3.0, respectively. The cells were harvested by centrifugation at 7,000g after 5 and 3.5 h of incubation for MazE_DR0416 and MazF_DR0417, respectively.

E. coli strain DH5α and BL21(DE3) were purchased from Nippon Gene (Tokyo, Japan) and were used for cloning and recombinant protein expression, respectively. A cloning vector, pTac-1, was purchased from BioDynamics (Tokyo, Japan). A modified pCold vector (TaKaRa, Shiga, Japan)^{47 (link)} was used for protein expression. Reagents were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) unless stated otherwise. Protein concentration was determined with a TaKaRa bicinchoninic acid (BCA) protein assay kit or TaKaRa Bradford protein assay kit.