Research on the new biotechnology for specific detection of environmental contamination by hazardous heavy metals.

20世纪以来，土壤或水中的重金属污染已成为工业化国家的一个严重环境问题。最近，这一问题在发展中国家被认为是一个非常危险的健康问题。为了预防和解决这一环境问题，我们致力于开发生物传感器技术，利用细菌细胞诱导的基因表达系统选择性地检测影响健康的重金属。(1)以细菌发光基因(Lux)为报告基因，建立了环境中有机汞化合物的检测体系：利用广谱耐汞细菌菌株大芽孢杆菌MB1的新的有机汞裂解酶基因merB‘s构建了merB3-荧光素酶(merB3-lux)转录融合载体。构建了编码同一巨型芽孢杆菌MB1操纵子/启动子序列和多聚体操纵子基因的另一个拟态粒，并将其作为反式基因表达载体与多聚体融合表达载体在Escherlchla coll细胞中表达。含有不同merB基因融合载体和1lux基因表达载体的转化子对烷基或芳香族有机汞的反应不同。(2)新型生物传感器可以检测到极低浓度的无机汞。用lux基因、merR和merT基因表达载体转化的细菌细胞显示Pm水平的汞污染。这种灵敏度比原子吸收法或离子耦合等离子体质谱等传统分析方法高出约一百倍。

Heavy metal contamination of soil or water has been frequent as a severe environmental problem in industrialized countries through th 20 century. Recently, this problem is recognized as a very dangerous health problem in developing countries. To prevent and solve this environmental problem, we aimed development of biosensor technology for selective detection of health affecting heavy metals using an induced gene expression system by bacterial cells. This research work was done to develop more sensitive heavy metal biosensors than conventional analytical methods.The two major results derived from this research project are as follows.(1) Using bacterial luminescence genes (lux) as reporter genes, we developed a detection system of organomercurial compounds in the environment: A new organomercury lyase genes, merB's, from a broad-spectrum mercury resistant bacterial strain, Bacillus magaterlum MB1, was used to construct merB3-luciferase (mer-lux) transcriptional fusion plasmids. Another piasmid encoding an operator/promoter sequence and mer operon genes from the same B. megaterlum MB1 was constructed and used as a transacting gene expression vector with the mer-lux fusion plasmids in a Escherlchla coll cell. The transformant with different merB genes fusion plasmids and a lux gene expression plasmids showed deferent response to alkyl-or aromatic-organomercurials tested in this study.(2) Very low concentration of inorganic mercury could be detected by newly developed biosensor devise bacterial cells. The bacterial cells transformed with lux genes, merR and merTgenes gene expression plasmids showed pM level mercury contamination. This sensitivity is about one hundred times higher than those of conventional analytical methods such as an atomic absorption method or an ion coupling plasma-mass spectrometory.