具超强富集U(Ⅵ)能力工程菌E.coli的构建

以鼠伤寒沙门氏菌基因组DNA为模板，PCR扩增得到非特异性酸性磷酸酶基因（phoN），将其克隆到pMD18T-Vector中。用Spe I、Nde I限制性内切酶对重组转移载体T-VectorphoN与穿梭载体pRADZ3分别进行双酶切，再将phoN片段和穿梭载体pRADZ3中的大片段通过T4DNA连接酶连接。经PCR与双酶切双重鉴定，证实重组穿梭载体pRADZ3phoN构建成功。转化Escherichia coli DH5α感受态细胞，使其在正常情况下表达PhoN蛋白，经Western blot 证实phoN基因在DH5α中成功表达。利用含pRADZ3phoN的工程菌进行富集U(Ⅵ)实验，结果表明该工程菌对U(Ⅵ)的富集量较宿主菌提高约4倍，达46.16mg/g，去除率为92.32%。

Construction of Uranium-superaccumulating Engineered E. coli

PhoN gene that was amplified from Salmonella enterica serovar Typhimurium genomic DNA by PCR, was cloned into pMD~(18) T-Vector. Recombined transfer vector T-VectorphoN was digested by restriction enzymes of Spe I and Nde I, and then the purified phoN gene was inserted into shuttle vector pRADZ3 which was digested by the same restriction enzymes. The recombined shuttle vector pRADZ3phoN was identified by PCR and restriction analysis, and transformed into E. coli DH5α competent cell. A recombinant fusion PhoN protein was expressed in normal growth condition without induction. The expression of PhoN protein in E. coli DH5α was confirmed by Western blot. The U (Ⅵ) bioaccumulation performance of engineered E. coli was evaluated. The results showed that the maximum U (VI) bioaccumulation capacity of engineered E. coli increased four times compared to original E. coli, reaching 46.16 mg/g, and the removal rate of U(Ⅵ) was 92.32%.