SBIR PHASE II: Biosensors for Trichloroethylene (TCE)

SBIR第二阶段项目涉及开发和测试生物传感器，以测量地下水中的三氯乙烯(TCE)。在第一阶段，通过将Mendocina假单胞菌KRL的TCE可诱导转录启动子融合到生物发光基因盒来构建TCE报告菌株。报告菌株在TCE存在的情况下产生光，产生的光量与存在的TCE量成正比。在第二阶段，我们将通过测量各种被三氯乙烯污染的地下水中的三氯乙烯来确定和验证生物传感器的性能特征。我们还将开发将报告菌株应用于TCE检测试剂盒的方法，包括使用冻干细胞。此外，我们将利用报告菌株开发在线生物传感器探针，用于生物反应器中TCE的连续监测和/或地下水监测井中TCE的井下监测。除了三氯乙烯生物传感器外，还将建造新的生物传感器来测量地下水中的汽油(BTEX)污染。BTEX传感器还将被用作质量保证/质量控制检查标准，以评估使用TCE生物传感器进行的TCE测量的准确性。通过用编码绿色荧光蛋白的基因取代生物发光报告盒，将构建一系列新的三氯乙烯和苯系物生物传感器，从而允许对用于模拟含水层中降解生物的迁移和表现的报告斑点进行显微镜检查。生物传感器和生物传感器探头将用于商业测量地下水中的三氯乙烯，改善生物反应器的过程控制，并降低就地修复技术的成本。

This Phase II SBIR project involves the development and testing of biosensors to measure trichloroethylene (TCE) in groundwater. During Phase I, TCE reporter strains were constructed by fusing a TCE-inducible transcriptional promoter from Pseudomonas mendocina KRl to a bioluminescence gene cassette. The reporter strains produced light in the presence of TCE, and the amount of light produced was proportional to the amount of TCE present. During Phase II we will determine and validate the performance characteristics of the biosensors by measuring TCE in a wide variety of TCE-contaminated groundwaters. We also will develop methods for applying the reporter strains in TCE test kits, including the use of freeze-dried cells. Furthermore, we will use the reporter strains to develop on-line biosensor probes that can be used for continuous monitoring of TCE in bioreactors and/or down hole monitoring of TCE in groundwater monitoring wells. In addition to TCE biosensors, new biosensors will be constructed to measure gasoline (BTEX) contamination in groundwater. The BTEX sensors will also be used as QA/QC check standards to evaluate the accuracy of TCE measurements made with the TCE biosensors. A new series of biosensors for both TCE and BTEX will be constructed by replacing the bioluminescence reporter cassettes with genes encoding the production of green fluorescent protein, thereby allowing microscopic examination of reporter stains used for modeling the migration and performance of degradative organisms in aquifers. The biosensors and biosensor probes will be used commercially to measure TCE in groundwater, improve process control in bioreactors, and reduce the cost of in situ remediation technologies.