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 wi...