SBIR PHASE I: Biosensors for Measuring Trichloroethylene (TCE) in Groundwater and Monitoring In Situ Bioremediation

这个小企业创新研究的第一阶段项目涉及开发用于测量地下水中三氯乙烯（TCE）和相关氯化溶剂的生物传感器。该项目涉及通过将报告基因与来自mendocina假单胞菌KR1和假单胞菌sp. ENVPC5的氯化溶剂诱导的甲苯氧化基因融合来创建全细胞生物传感器。各种报告基因将被评估，但工作将集中在使用生物发光基因，已广泛用于生物传感器的创建。最初的生物传感器开发将涉及在报告基因和克隆的甲苯氧化结构基因之间创建转录融合。一旦产生，融合体将通过标记交换转移回原始宿主生物体。另外的生物传感器将通过克隆整个甲苯氧化途径的规则到大肠杆菌中，并插入报告基因，使其在对TCE的反应中表达。这种方法将允许评估最适合用作生物传感器的宿主生物。研究人员将测试这些生物传感器对地下水中TCE的敏感性，并将报告基因的表达与野生型甲苯氧化基因对TCE的响应进行比较。这些生物传感器最终将用于开发在线传感器，用于监测地下水中的TCE，开发TCE测试试剂盒，以及评估TCE降解微生物的原位性能。第一阶段的研究将导致活体TCE生物传感器的发展，可用于构建在线监测设备和测试套件。它们还将有助于评估、监测和模拟原位tce降解细菌的性能。开发一种廉价的生物传感器系统将降低常规化学监测的成本，允许在线监测，并提高环境修复的成本效益。＊*＊

*** 9560186 Steffan This Small Business Innovation Research Phase I project involves the development of biosensors for measuring trichloroethylene (TCE) and related chlorinated solvents in groundwater. The project involves creating whole-cell biosensors by fusing reporter genes with chlorinated solvent-induced toluene oxidation genes from Pseudomonas mendocina KR1 and Pseudomonas sp. ENVPC5. A variety of reporter genes will be evaluated, but the work will focus on the use of bioluminescence genes that have been widely used for the creation of biosensors. Initial biosensor development will involve creating transcriptional fusions between the reporter genes and cloned toluene oxidation structural genes. Once created, the fusions will be transferred back to the original host organism via marker exchange. Additional biosensors will be created by cloning the entire regulons of the toluene oxidation pathways into E. coli and inserting the reporter genes so it is expressed in response to TCE. This approach will allow an evaluation of the most appropriate host organisms for use as biosensors. The biosensors will be tested for their sensitivity for measuring TCE in ground water, and the expression of the reporter genes will be compared to expression of the wild-type toluene oxidation genes in response to TCE. The biosensors will ultimately be used for developing on-line sensors for monitoring TCE in groundwater, for developing TCE test kits, and for evaluating the in situ performance of TCE-degrading microorganisms. This Phase I research will result in the development of living TCE biosensors that can be used to construct on-line monitoring devices and test kits. They will also aid in evaluating, monitoring and modeling the performance of TCE-degrading bacteria in situ The development of an inexpensive biosensor system will reduce the cost of routine chemical monitoring, allow on-line monitoring, and increase the cost effectiveness of environmental remediation. ***