Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription

Synthetic biology has enabled the development of powerful nucleic acid diagnostic technologies for detecting pathogens and human health biomarkers. Here we expand the reach of synthetic biology-enabled diagnostics by developing a cell-free biosensing platform that uses RNA output sensors activated by ligand induction (ROSALIND) to detect harmful contaminants in aqueous samples. ROSALIND consists of three programmable components: highly-processive RNA polymerases, allosteric transcription factors, and synthetic DNA transcription templates. Together, these components allosterically regulate the in vitro transcription of a fluorescence-activating RNA aptamer: in the absence of a target compound, transcription is blocked, while in its presence a fluorescent signal is produced. We demonstrate that ROSALIND can be configured to detect a range of water contaminants, including antibiotics, toxic small molecules, and metals. Our cell-free biosensing platform, which can be freeze-dried for field deployment, creates a new capability for point-of-use monitoring of molecular species to address growing global crises in water quality and human health.