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

* 9560186斯蒂芬 该小企业创新研究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门多萨假单胞菌KR 1和假单胞菌ENVPC 5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。 将对各种报告基因进行评估，但工作将集中在生物发光基因的使用上，这些基因已被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创建报告基因和克隆的甲苯氧化结构基因之间的转录融合。 一旦产生，融合体将通过标记交换转移回原始宿主生物体。 通过将甲苯氧化途径的整个调节子克隆到大肠杆菌中，将产生更多的生物传感器。大肠杆菌中，并插入报告基因，使其表达响应TCE。 这种方法将允许评价最合适的宿主生物体用作生物传感器。 将测试生物传感器测量地下水中TCE的灵敏度，并将报告基因的表达与响应TCE的野生型甲苯氧化基因的表达进行比较。 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三氯乙烯，开发三氯乙烯检测试剂盒，以及评估三氯乙烯降解微生物的原位性能。 第一阶段的研究将导致开发活的TCE生物传感器，可用于构建在线监测设备和检测试剂盒。 它们还将有助于评估，监测和模拟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. ***