There is an on-going need for simple, rapid and cost-effective analytical methods capable of detecting toxicsubstances in the environment and in biological samples. Among these toxic compounds, polychlorinatedbiphenyls (PCBs) have been extensively studied and proven to be harmful to humans and the environment,and consequently classified as Superfund chemicals. More recently, hydroxylated PCBs (OH-PCBs) havealso been more closely examined as potentially exhibiting significant toxic health effects. Due to the toxicityand environmental and biological persistence of PCBs and OH-PCBs, it is necessary to have efficient andeconomical methods to detect and quantify them. Currently employed traditional analytical methods arecostly, time- and labor-consuming, not amenable to field analyis, and not appropriate for extensivemonitoring of target analytes in biological samples. To that end, the long-term objective of this proposal is todesign and develop molecular biosensors, employing genetic engineering tools, which are selective,sensitive, portable, and inexpensive, and propose them as a viable alternative to traditional analyticalmethods for the detection of PCBs and OH-PCBs in biological and environmental samples. Specifically,whole-cell biosensors will be constructed that are based on bacteria engineered to harbor a plasmidcontaining the genes encoding for the proteins involved in the catabolic pathways of Superfund chemicalscoupled to those of a signal-producing reporter protein. Additionally, the regulatory proteins of bacterialresistance operons will be redesigned to improve their binding selectivity to target analytes, and will beincorporated as the recognition elements in a new biosensing format. These redesigned proteins should beable to act like molecular switches to detect the presence of the target compounds in samples. Furthermore,the newly developed biosensing systems will be miniaturized and integrated into enabling technologies for insitu detection and field studies. As part of the enabling technologies, the transformation of bacterial wholecellsensors into spores, as long-term, highly rugged storage and transport elements, will be explored.These spore-based biosensors, as well as the molecular switches, will then be incorporated into portableminiaturized microfluidic platforms for field sensing. These biosensing systems provide with new tools formonitoring of human health and the environment, therefore, they are of high relevance for public health.
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