Targeted inactivation of argA in E. coli and mutant complementation by polN. (A) Representational map for construction of the CH3 mutant. (B) Identification of CH2 mutants. As 1.1-kb argA fragment was replaced by 1.0-kb aac(3)IV gene, the PCR product for CH3 mutants was ca. 1.5-kb, while the wild type produces 1.6-kb PCR product. (C) Complementation of CH3 mutant by polN. CH3 mutant of E. coli BL21(DE3) (1) and CH3 mutant containing pET28a (2) were used as negative controls, while CH3 mutant containing pJTU2838 with inserted polN was indicated as (3). The final concentration for arginine and thymidine used in this experiment is 50 μg/ml and 200 μg/ml, respectively, and the liquid cultures were incubated at 37°C for 90 h.