Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

Abstract

Advances in biosensor engineering have enabled the design of programmable molecular systems to detect a range of pathogens, nucleic acids, and chemicals. Here, we engineer and field-test a biosensor for fluoride, a major groundwater contaminant of global concern. The sensor consists of a cell-free system containing a DNA template that encodes a fluoride-responsive riboswitch regulating genes that produce a fluorescent or colorimetric output. Individual reactions can be lyophilized for long-term storage and detect fluoride at levels above 2 parts per million, the EPA's most stringent regulatory standard, in both laboratory and field conditions. Through onsite detection of fluoride in a real-world water source, this work provides a critical proof-of-principle for the future engineering of riboswitches and other biosensors to address challenges for global health and the environment.

Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins

“…While CFPS conditions may vary according to the unique transcription/translation requirements for the synthesis of individual proteins, these reactions can be tailored by optimizing concentrations of the CFPS reagents. As such, CFPS has enabled a wide range of applications in directed evolution, synthetic biology, glycoscience, metabolic engineering, and education (Des Soye, Gerbasi, Thomas, Kelleher, & Jewett, 2019; Harris & Jewett, 2012; Karim & Jewett, 2016; Karim et al, 2020; Kightlinger et al, 2019; Lin et al, 2020; Martin et al, 2018; Silverman et al, 2020; Stark et al, 2019; Thavarajah et al, 2020). LETs offer a unique advantage over their circular counterparts because extensive plasmid construction steps are not required.…”

Applications, challenges, and needs for employing synthetic biology beyond the lab

“…Several groups have demonstrated proof-of-concept field deployment of other kinds of cell-free sensing platforms, a portable colorimetric detection platform for sensing of endocrinedisrupting chemicals using cell-free protein synthesis of an allosterically activated fusion protein 110 , as well as a riboswitchbased sensor for colorimetric detection of fluoride contamination in groundwater samples which was capable of detecting concentrations around 2 ppm (the EPA limit) 111 . Jung et al 112 demonstrated detection of a wide range of common water contaminants in municipal water samples including small molecules, metals, and antibiotics using RNA Output Sensors Activated by Ligand Induction (ROSALIND), a sensing platform employing allosteric regulation of in vitro transcription of fluorescence-activated RNA aptamers.…”

“…All three of these platforms were demonstrated to be lyophilizable and shelf stable for at least 2.5 months. In addition, they all require 37°C incubation which has been demonstrated to be achievable using a low-cost handwarmer as well as by using a patient's own body temperature 111 . Overall, the field-ready features demonstrated by these platforms are quite promising in terms of enabling their deployment in settings with limited or no power.…”

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

“…Cell-free biosensing is emerging as a low-cost, easy-to-use and field-deployable diagnostic technology that can be applied to detect a range of contaminants related to human and environmental health [1][2][3]. At their core, these systems consist of two layers: a sensing layer that includes an RNA or protein-based biosensor and an output layer that includes a reporter construct.…”