Design and Optimization of a Cell-Free Atrazine Biosensor

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 component ratios of 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 modularly designed, which should enable this work to serve as an effective proof-of-concept for rapid fielddeployable detection of complex organic water contaminants.

A primer on emerging field-deployable synthetic biology tools for global water quality monitoring

“…Both whole-cell and cell-free biosensors have previously been used to detect metals by utilizing natural or engineered proteins; sensors have been reported for cadmium, lead, mercury, arsenic, copper, zinc, nickel, and cobalt, with sensitivities ranging from low parts per million to parts per billion 25,31,82,83 . Sensors for atrazine, a toxic herbicide, have also been developed by encoding a natural metabolic pathway for atrazine's conversion to cyanuric acid, which can be detected with a known protein sensor 84,85 . Furthermore, new cell-free approaches can detect a range of PPCPs, including multiple families of antibiotics and benzalkonium chloride 25,82,86 .…”

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

“…A recent field example is the use of a cell-free biosensor to detect the environmental toxin cyanuric acid (CYA) in water through coupling E. coli gene expression machinery with a non-native transcriptional regulator from Pseudomonas sp., via engineering of hybrid Pseudomonas-E. coli promoters capable of functioning in a freeze-dried E. coli cell-free system 93 . To expand the collection of detectable analytes beyond nucleic acids and a specific set of well-characterized chemical contaminants, Silverman et al 94 developed a platform for organic molecule detection via coupling metabolic conversion of target molecules with transcription-factor-based biosensing for efficient detection of atrazine. This modular platform enabled multiplexed sensing all within one mixture via the addition of multiple cell-free extracts with specific reporter plasmids.…”

Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

“…TF‐based cell‐free biosensors have been used to identify specific small molecules, such as inorganic ions and organic molecules (bacterial quorum sensing molecules), and they are also used in high‐throughput screening and metabolic engineering. [ 48,60,61 ] Specific TFs or natural conformational TFs are selected by different analytes, such as TF in response to aromatic compounds (XylS‐AraC, XylR‐NtrC, and LysR), metal ions (MerR, ArsR, DtxR, Fur, and NikR), or antibiotics (TetR and MarR). [ 62 ] When analytes (ligands) are present in a cell‐free environment, the response of TFs is immediately obtained, and binding of ligands to their activated TFs or the release of ligand‐bound inhibitory TFs can lead to the reporter gene expression ( Figure 2 A).…”

“…For unknown ligands, they can be converted into detectable ligands by using metabolic enzymes. [ 18,48 ]…”

“…[ 102 ] Silverma et al. [ 48 ] developed a cell‐free biosensor to detect the herbicide atrazine. They combined that CYA (cyanuric acid) cell‐free sensor with a reconstituted cell‐free metabolic pathway that converts atrazine to CYA through three steps in a one‐pot reaction.…”