FIGURE 2.

Schematic procedures for the production of MazF-bs(arg) and MazF-bs(can). A, DNA sequence of the mazF-bs gene. The gene is designed to be ACA-less and codon-optimized for E. coli. The amino acid sequence of MazF-bs is shown under the DNA sequence. B, dual inducible SPP system. The BL21(DE3) (ΔargHΔtrpCΔhisB) cells were transformed with pColdIIImazF-bs together with pACYCmazF(ΔH) and grown in a 1-liter culture of M9-glucose medium in the presence of Arg (20 μg/ml), His (20 μg/ml), and Trp (20 μg/ml) at 37 °C. When the A₆₀₀ value reached 0.5, the culture was chilled in an ice-water bath for 5 min and incubated at 15 °C for 1 h to acclimate the cells to cold shock conditions. Cells were harvested and washed twice with M9 medium. The cells were resuspended in 50 ml of M9-glucose medium containing Arg (20 μg/ml) and Trp (20 μg/ml) but without His. IPTG (0.5 mm) was added to induce the only expression of MazF(ΔH) followed by an additional 2-h incubation at 15 °C. Cells were harvested and washed twice with M9 medium. The cells were resuspended in 50 ml of M9-glucose medium containing His (20 μg/ml), and Trp (20 μg/ml), Can (100 μg/ml), and IPTG (0.5 mm) to incorporate Can into MazF-bs. The cell culture was incubated at 15 °C for additional 24 h to induce MazF-bs(can). When Arg (20 μg/ml) was added instead of Can, MazF-bs(arg) was produced.