Sensing Superfund Chemicals with Recombinant Systems

检测超级基金化学品是从环境中消除它们或研究它们对生物过程的影响的第一步。为此，我们建议开发分子工具，用于高灵敏和直接检测超级基金化学品，如多氯联苯。这些分子工具将包括基于全细胞和基于蛋白质的光学传感系统。为此，我们建议利用在多氯联苯存在下诱导的自然可用操纵子和参与其降解的分解代谢酶。我们计划通过加入报告基因来修饰这些操纵子，这将使我们能够监测它们在多氯联苯存在的情况下的诱导。参与多氯联苯降解的分解代谢酶，特别是联苯双加氧酶(BphAt)，将被分离并标记信号转导部分，用于分析这些化合物。此外，为了提高这些分解代谢酶的选择性 针对多氯联苯的同系物，提出了旨在修饰酶活性部位的诱变研究。为了使这些生物传感系统能够进行现场研究和高通量样品分析，我们建议将它们整合到一个微流控平台中。这个小型化的分析平台应该会在超级基金化学品的现场监测中得到应用。具体地说，我们计划将我们的多氯联苯生物传感系统改造为光盘离心式微流控平台。我们建议的生物传感系统的优势包括灵敏度、便携性以及分析不同基质(如土壤、水和生物样品)中多氯联苯的可行性。

Detection of Superfund chemicals is a first step towards eliminating them from the environment or studying their effect on biological processes. To that end, we propose to develop molecular tools for the highly sensitive and direct detection of Superfund chemicals such as PCBs. These molecular tools will include whole cell- and protein-based optical sensing systems. For that, we propose to utilize naturally available operons induced in the presence PCBs and the catabolic enzymes involved in their degradation. We plan to modify these operons by incorporating the gene for the reporter, which will allow us to monitor their induction in the presence of PCBs. Catabolic enzymes involved in the degradation of PCBs, in particular the enzyme biphenyl dioxygenase (BphAt), will be isolated and labeled with signal transduction moieties for the analysis of these compounds. In addition, to improve the selectivity of these catabolic enzymes towards PCB congeners, mutagenesis studies aimed at modifying the active site of the enzyme are proposed. In order to make these biosensing systems amenable to field studies and high throughput sample analysis, we propose to incorporate them into a microfluidics platform. This miniaturized analytical platform should find applications in the on-site monitoring of Superfund chemicals. Specifically, we plan to adapt our biosensing systems for PCBs to a compact disc centrifugal microfluidics platform. The advantages of our proposed biosensing systems include sensitivity, portability, and the feasibility of analysis of PCBs in different matrices, such as soil, water, and biological samples.