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.
Keywords: TX-TL; atrazine; biosensor; cell-free; cyanuric acid; metabolism; synthetic biology; transcription factor.