Metagenomic Discovery of Water Contaminant Biosensors

OUSD (R&E) CRITICAL TECHNOLOGY AREA(S): Chem Bio Defense; Sensors; Biotechnology OBJECTIVE: Develop and employ a metagenomic pipeline to discover novel biosensors for water contaminants of DoD concern. Biosensors can be further engineered or evolved to enable in-field sensing of these contaminants. DESCRIPTION: Clean drinking water is an essential resource for human survival and the ability to detect harmful contaminants in drinking water is critical for human health and safety. Many contaminants entering water sources through natural processes or human activity are cause for concern due to their toxicological effects or ability to persist in the environment. Access to clean water and methods to assess water quality are particularly challenging in field-forward and resource-poor environments; indeed, drinking water presents a huge logistical burden for deployed troops with a heavy reliance on bottled water [1,2]. Microorganisms constantly monitor their environment and have naturally evolved mechanisms to sense and respond to nearly any environmental input. Microorganisms provide a rich resource for potential naturally-derived sensors against a multitude of threats and are being harnessed to create whole-cell and cell-free biosensors [3,4], such as biosensors to detect heavy metal contamination in water [5,6]. A key limitation, however, is the lack of well-characterized, specific, and adaptable biosensors available to convert environmental signals into biological signals for downstream processing. This dearth of well-characterized biosensors currently hampers products such as paper-based cell-free sensors [7] that have great potential as a highly-fieldable, broad-spectrum detection technology to protect the Warfighter. While it is known that cells naturally respond to broad-spectrum analytes, the specific mechanisms remain obscure outside of a handful of model biosensors; thus, discovery of novel biosensors through identification of natural sensors and downstream engineering is desired. A metagenomic pipeline for biosensor discovery would greatly facilitate the development of new sensors and ultimately provide a new generation of sensing technology to the Warfighter. The objective of this topic is to develop and employ a metagenomic screening approach for biosensor discovery of water contaminants of DoD concern and to further develop and evaluate these biosensors for improved performance and portability into a biology-based system with fieldability potential (e.g., cell-free expression system). These biosensors must be implementable in a molecular circuit to convert chemical signals to downstream biological signals. Of particular interest are protein-based transcription factors that transduce a signal via promoter regulation in response to the presence of the analyte of interest; however, other mechanisms may also be considered. The end goal for this topic is the development of functional biosensors for multiple water contaminants of DoD concern with future biosensor discovery enabled by the metagenomic screening pipeline developed and implemented under this effort. PHASE I: This topic is for Phase I submission only. The Department of the Army will accept Phase I proposals for the cost of up to $250,000 for a 6-month period of performance. Develop a metagenomic pipeline for discovery of novel biosensors. Demonstrate this pipeline for discovery of biosensors against three relevant water contaminant targets that do not have existing biosensors in the literature. TB MED 577 [8] and Technical Guide 230 [9] are two Army documents containing lists of water contaminants of concern. Of particular interest are per- and polyfluoroalkyl substances (PFAS), toxins, and uranium. PHASE II: Further engineer or evolve the three biosensors developed in Phase I for improved sensor performance (e.g., signal-to-noise ratio, sensitivity, specificity). Demonstrate portability of the sensors into a cell-free or cell-based system (cell-free protein expression system preferred). Expand the metagenomic biosensor discovery approach for three additional targets in coordination with DoD scientists (e.g., opioids/fentanyl, explosives/RDX, chromium, selenium). PHASE III DUAL USE APPLICATIONS: A robust water quality sensing platform would have considerable impact both for DoD field-forward operations as well as for civilian and humanitarian water quality management. Commercial use cases include at-home water testing, municipal water testing, and water quality testing to support humanitarian efforts around the world. In recent years, there has been increased civilian concern regarding water quality as instances of water containing lead, PFAS, or other harmful contaminants have been increasingly reported. The technology can also be used for applications beyond water quality, such as pathogen identification and pandemic response. REFERENCES: Charkoudian N, Kenefick RW, Lapadula AJ, Swiston AJ, Patel T, Blanchard LA, Caruso EM, Luippold AJ, Cheuvront SN. Planning Military Drinking Water Needs: Development of a User-Friendly Smart Device Application. Mil Med. 2016, 181(9):1142-50. Moore, JS. The U.S. Military's reliance on bottled water during military operations. Masters Thesis, National Defense University. 2011. Moraskie M, Roshid MHO, O'Connor G, Dikici E, Zingg JM, Deo S, Daunert S. Microbial whole-cell biosensors: Current applications, challenges, and future perspectives. Biosens Bioelectron. 2021, 191:113359. Lee KH, Kim DM. In Vitro Use of Cellular Synthetic Machinery for Biosensing Applications. Front Pharmacol. 2019, 10:1166. Beabout K, Bernhards CB, Thakur M, Turner KB, Cole SD, Walper SA, Ch vez JL, Lux MW. Optimization of Heavy Metal Sensors Based on Transcription Factors and Cell-Free Expression Systems. ACS Synth Biol. 2021, 10(11):3040-3054. Bereza-Malcolm LT, Mann G, Franks AE. Environmental sensing of heavy metals through whole cell microbial biosensors: a synthetic biology approach. ACS Synth Biol. 2015, 4(5):535-46. Pardee K, Green AA, Ferrante T, Cameron DE, DaleyKeyser A, Yin P, Collins JJ. Paper-based synthetic gene networks. Cell. 2014, 159(4):940-54. TB MED 577 Sanitary Control and Surveillance of Field Water Supplies. U.S. Department of the Army. 2010. Technical Guide 230 Environmental Health Risk Assessment and Chemical Exposure Guidelines for Deployed Military Personnel. U.S. Army Public Health Command. 2013. KEYWORDS: Biosensor; metagenomics; transcription factors; cell-free systems; synthetic biology; water quality; water contaminants; PFAS