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

环境有害重金属污染特异性检测的新型生物技术研究

• 批准号: 13450216
• 负责人: ENDO Ginro
• 金额: $ 9.66万
• 依托单位: Tohoku Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13450216/
• 关键词: heavy metal contamination; biosensor; gene expression system; bio-luminescence gene; mercury resistance operon; quantitative analysis technology; 分析定量技術

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.

整个20世纪，土壤或水的重金属污染已成为工业化国家经常出现的严重环境问题。最近，这个问题被认为是发展中国家非常危险的健康问题。为了预防和解决这一环境问题，我们的目标是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检测影响健康的重金属。本研究工作的目的是开发比传统分析方法更灵敏的重金属生物传感器。该研究项目得出的两个主要结果如下：（1）利用细菌发光基因（lux）作为报告基因，我们开发了环境中有机汞化合物的检测系统：来自广谱抗汞菌株Bacillus magaterlum的新有机汞裂解酶基因merB's MB1，用于构建merB3-荧光素酶（mer-lux）转录融合质粒。构建了编码来自相同巨大芽孢杆菌MB1的操纵子/启动子序列和mer操纵子基因的另一个质粒，并在大肠杆菌细胞中与mer-lux融合质粒一起用作反式基因表达载体。具有不同merB基因融合质粒和lux基因表达质粒的转化子对本研究中测试的烷基或芳香族有机汞表现出不同的反应。(2)新开发的生物传感器装置细菌细胞可以检测到极低浓度的无机汞。用lux基因、merR和merTgenes基因表达质粒转化的细菌细胞显示出pM水平的汞污染。该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约一百倍。

项目成果

T.Yamagata, M.Ishii, M.Narita, C.C.Huang, G.Endo: "Bio-affecting mercury detection using mercury resistance gene module fused with bioluminescence reporter genes"Proceedings of Asian Waterqual 2001-First IWA Asia-Pacific Regional Conference. Vol.1. 329-33

T.Yamagata、M.Ishii、M.Narita、C.C.Huang、G.Endo：“使用与生物发光报告基因融合的耐汞基因模块进行生物影响汞检测”亚洲水质量 2001 年第一届 IWA 亚太地区会议论文集。

G.Endo(共著): "Biogeochemical Aspect of Earth System and Bioremediation of Polluted Environments(T.Hattori Ed.)"OECD-ISEB16, ISBN4-9901886-0-8. 124 (2003)

G. Endo（合著者）：“地球系统的生物地球化学方面和污染环境的生物修复（T. Hattori Ed.）” OECD-ISEB16，ISBN4-9901886-0-8（2003）。

山肩健史, 石井秀学, 成田勝, 熊谷康, 黄介辰, 遠藤銀朗: "新たに発見された有機水銀分解遺伝子merB3とその発現調節系を利用した有機水銀の検出方法に関する研究"水環境学会誌. 24. 219-224 (2001)

Takeshi Yamashita、Hidenari Ishii、Masaru Narita、Yasushi Kumagai、Keishin Oh、Ginro Endo：“利用新发现的有机汞分解基因merB3及其表达调控系统进行有机汞检测方法的研究”水环境协会杂志24. 219。 -224 (2001)

T.Yamagata, M.Ishii, M.Narita, C.C.Huang, G.Endo: "Bio-affecting mercury detection using mercury resistance gene Module fused with bioluminescence reporter genes."Water Science and Technology. 46. 253-256 (2002)

T.Yamagata、M.Ishii、M.Narita、C.C.Huang、G.Endo：“使用与生物发光报告基因融合的耐汞基因模块进行生物影响汞检测。”水科学与技术。

G.Endo, M.Narita, C-C.Huang, S.Silver: "Microbial heavy metal resistance transposons and plasmids: Potential use for Environmental Biotechnology."J.Environmental Biotechnology. Vol.2, No.2. 71-82 (2002)

G.Endo、M.Narita、C-C.Huang、S.Silver：“微生物重金属抗性转座子和质粒：环境生物技术的潜在用途。”J.环境生物技术。