Abstract
Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the ratios of enriched extracts in the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modular. Our work thus serves as an effective proof-of-concept for a scheme of "metabolic biosensing", which should enable rapid, field-deployable detection of complex organic water contaminants.
Highlights
IntroductionCell-free gene expression (CFE) has recently emerged as a powerful strategy for rapid, field-deployable diagnostics for nucleic acids and chemical contaminants
Due to the burden imposed by synthesizing several proteins in situ in a single batch Cell-free gene expression (CFE) reaction, we developed an extract mixing strategy, where individual extracts enriched with a single enzyme or transcription factor are combined to reconstitute the complete biosensing reaction
BL21 E. coli, lysed and prepared into individual extracts, which can be mixed in fixed ratios to optimize detection of atrazine. (B) Cell-free detection of atrazine requires the presence of all three pathway enzymes. (C) The sensor is capable of detecting cyanuric acid and atrazine, as well as propazine, a triazine of similar chemical structure, but discriminates against melamine
Summary
Cell-free gene expression (CFE) has recently emerged as a powerful strategy for rapid, field-deployable diagnostics for nucleic acids and chemical contaminants.. Cell-free gene expression (CFE) has recently emerged as a powerful strategy for rapid, field-deployable diagnostics for nucleic acids and chemical contaminants.6-9 One reason for this success is that CFE reactions minimize many of the constraints of whole-cell sensors, including mass transfer barriers, cytotoxicity, genetic instability, plasmid loss, and the need for biocontainment.. Previously reported cell-free biosensors have so far been limited to detecting either nucleic acids or chemical contaminants that can be directly sensed with well-characterized allosteric protein transcription factors or riboswitches.. We expand the ability of cell-free biosensors to detect complex organic molecules by developing a combined metabolism and biosensing strategy. A range of new CFE-based diagnostics could be developed by combining in vitro metabolic conversion with natural transcriptional biosensors
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