A Paper-Based Synthetic Biology Platform for the On-Demand Testing of Water Quality

安全饮用水对公共健康至关重要，但日益受到人为活动的威胁， 老化的基础设施，重金属和其他毒素污染它。在超级基金附近尤其如此 和布朗菲尔德场地，已知或怀疑工业活动已导致水污染 源最值得注意的是饮用水被铅和砷污染，这可能导致铅中毒。 中毒和砷中毒，并可导致发育障碍，身体异常， 和癌症一旦发现，这些污染物可以通过现有的净化技术来减轻。 然而，由于最近发生的事件，如密歇根州弗林特或新泽西州纽瓦克的危机， 水管理和过滤故障正在导致重大的公共卫生威胁。解决方案的一部分 安全用水管理面临的挑战是经常进行水质检测。然而，可靠的测试仍然有限 成本高、耗时且需要大量实验室的分析化学技术 基础设施和技术专长。这使得解决关键水问题所需的大规模测试变得复杂 管理问题，并且一直是消费者对供水进行常规检测的一大障碍。这里 我们建议通过开发一个新的技术平台来解决这些问题，该平台将允许可靠的、低成本的 成本、现场和按需监测供水中的有害污染物。我们的技术 合成生物学的最新创新允许重新利用天然的变构转录因子， 它可以感知特定的毒性配体，如重金属，并通过激活基因表达做出反应。的 支持基因表达过程和可见基因表达的无细胞合成生物学反应的应用 报告子允许组装“无细胞生物传感器”，其是可以冷冻干燥用于 长期储存和简单分配。这些传感器与水样品的再水合然后激活 在有毒化合物的存在下，反应并产生可检测的信号。本第一阶段提案详细说明了 一系列互补的目标，以实现提高特异性和灵敏度的生物传感平台， 检测铅和砷作为具有重大健康问题的模型目标污染物的背景， 将其结合到适合消费者使用的方便的基于纸的格式中。我们的方法包括 开发和应用生物信息学方法鉴定天然存在的转录因子 具有改进的性能特征的同源物，高通量无细胞合成生物学方法 快速表征其性能，以及嵌入和测试这些传感器的新制造技术 在纸基基底上。这一建议的成功结果将导致一个多路复用，基于纸张的设备 这将解决可靠和负担得起的水质监测污染物铅的问题， 砒霜这项工作将建立这种技术的概念验证，使第二阶段的目标， 在实际水样中实现用户指导的产品规格，并扩大生产规模。

Safe drinking water is essential for public health, yet is increasingly threatened by anthropogenic activities and aging infrastructure that contaminate it with heavy metals and other toxins. This is especially true near Superfund and brownfield sites, where industrial activity is either known or suspected to have resulted in pollution of water sources. Most notable is the contamination of drinking water with lead and arsenic, which can result in lead poisoning and arsenicosis, respectively, and can contribute to developmental disorders, physical abnormalities, and cancer. Upon discovery, these contaminants can be mitigated by existing purification technologies. However, as recent events such as the crises in Flint, MI or Newark, NJ exemplify, the combination of improper water management and filtration failure is leading to major public health threats. Part of the solution to the challenge of safe water management is frequent water quality testing. However, reliable testing remains limited to analytical chemistry techniques that are costly, time consuming, and require substantial laboratory infrastructure and technical expertise. This complicates the large scale testing needed to address critical water management issues, and has been a large barrier for the routine testing of water supplies by consumers. Here we propose to address these issues by developing a new technology platform that will allow for the reliable, low- cost, on-site and on-demand monitoring of harmful contaminants within water supplies. Our technology is built from recent innovations in synthetic biology that allow the repurposing of natural allosteric transcription factors that can sense specific toxic ligands, such as heavy metals, and respond by activating gene expression. The use of cell-free synthetic biology reactions that support gene expression processes and visible gene expression reporters allows the assembly of “cell-free biosensors”, which are in vitro reactions that can be freeze-dried for long term storage and simple distribution. Rehydration of these sensors with a water sample then activates the reaction and produces a detectable signal in the presence of a toxic compound. This Phase I proposal details a series of complementary aims for achieving improved specificity and sensitivity of this biosensing platform, in the context of detecting lead and arsenic as model target contaminants of significant health concern, and incorporating it within a convenient paper-based format suitable for consumer use. Our approach includes the development and application of bioinformatic approaches to identify naturally-occurring transcription factor homologues with improved performance characteristics, high throughput cell-free synthetic biology approaches to rapidly characterize their performance, and new manufacturing techniques to embed and test these sensors on paper-based substrates. A successful outcome of this proposal will lead to a multiplexed, paper-based device that will address the problem of reliable and affordable water quality monitoring for the contaminants lead and arsenic. This work will establish a proof-of-concept of this technology that will enable the Phase II goals of achieving user-guided product specifications in real-world water samples and manufacturing scale-up.