Tyler Lucci

Overuse of pesticides and herbicides, such as glyphosate, is a major global concern due to potential negative impacts on human and environmental health. This makes rapid detection of these environmental chemical contaminants necessary for monitoring and mitigation efforts. Our previously developed RNA output sensor activated by a ligand induction (ROSALIND) cell-free biosensor platform enables rapid, point-of-need detection of chemicals through transcription factors that sense chemical targets and activate reporter gene expression in vitro. However, ROSALIND currently relies on known transcription factors that directly bind chemical ligands to transduce a signal, limiting the range of chemicals that can be detected by this technology. Here, we expand ROSALIND to incorporate enzymes that convert chemical targets of interest into detectable compounds. We show a proof-of-principle of this design by engineering ROSALIND to detect the herbicide glyphosate by enzymatically converting it to glyoxylate, which binds the transcription factor AllR to activate the gene expression. This work develops a workflow for expanding in vitro transcription biosensors by interfacing them with enzymatic conversion pathways and lays the groundwork for developing functional glyphosate sensors for real-world applications.

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds, such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including the ability to be assembled, freeze-dried, distributed, and then used at the point of need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid-handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semiautomated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride-sensing cell-free biosensors and demonstrate that they perform close to expected detection outcomes.

Water contamination by harmful chemicals is a growing global concern, creating a need to develop technologies that can detect a range of target compounds at the required concentrations. Here, we address this need by merging biological allosteric transcription factors with DNA-coated nanomechanical microcantilevers to detect chemicals in water with a digital readout. After proof-of-concept demonstration and optimization to detect anhydrotetracycline with the TetR transcription factor, we use the CadC transcription factor to detect Pb²⁺ and Cd²⁺ in water at concentrations down to 2 and 1 ppb, respectively, in less than 15 min. A computational model suggests this improvement in sensitivity could be achieved by the DNA-coated microcantilever surface changing the transcription factor binding properties. Our findings demonstrate a promising approach for water quality monitoring with fast, highly sensitive, digital readout.

Cell-free systems are powerful synthetic biology technologies that can recapitulate gene expression and sensing without the complications of living cells. Cell-free systems can perform more advanced functions when genetic circuits are incorporated. Here we expand cell-free biosensing by engineering a highly specific isothermal amplification circuit called polymerase strand recycling (PSR), which leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We first construct simple PSR circuits to detect different RNA targets with high specificity. We then interface PSR circuits to amplify signals from allosteric transcription factor-based biosensors for small molecule detection. A double equilibrium model of transcription factor–DNA/ligand binding predicts that PSR can improve biosensor sensitivity, which we confirm experimentally by improving the limits of detection by 10-fold to submicromolar levels for two biosensors. We believe this work expands the capabilities of cell-free circuits and demonstrates PSR’s potential for diverse applications in biotechnology.

There is growing concern around the negative health impacts associated with contamination of drinking water by harmful chemicals. Technology that enables fast, cheap, and easy detection of ions and small molecules in drinking water is thus important for reducing the incidence of these negative health impacts. Here, we describe a sensor for detecting Cu²⁺ in water that provides colorimetric results in 15 min or less and functions in a just-add-water format. The sensor contains cheap reagents including salts, buffer, oxidant, chromogen, surfactant, and optionally a chelating agent. The sensor is assembled and lyophilized for shelf-stability and field-deployment. Rehydrating the sensor with water containing Cu²⁺ results in chromogen oxidation and blue color formation to visually indicate the presence of Cu²⁺. The sensor demonstrates high selectivity toward Cu²⁺ against other metal cations, functionality in field samples, shelf-stability, and can be tuned to activate at different Cu²⁺ threshold concentrations. This sensor thus has the potential to meet a variety of needs, such as point-of-need testing for Cu²⁺ to ensure water supplies meet health guidelines, such as the United States Environmental Protection Agency's Lead and Copper Rule.

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

Cell-free systems are powerful synthetic biology technologies because of their ability to recapitulate sensing and gene expression without the complications of living cells. Cell-free systems can perform even more advanced functions when genetic circuits are incorporated as information processing components. Here we expand cell-free biosensing by engineering a highly specific isothermal signal amplification circuit called polymerase strand recycling (PSR) that leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We develop design rules for PSR circuit components and use these rules to construct modular biosensors that can directly sense different RNA targets with limits of detection in the nM range and high specificity. We then use PSR for signal amplification within allosteric transcription factor-based biosensors for small molecule detection. We use a double equilibrium model of transcription factor:DNA and transcription factor:ligand binding interactions to predict biosensor sensitivity enhancement by PSR, and then demonstrate this approach experimentally by achieving 3.6-4.6-fold decreases in biosensor EC50 to sub micromolar ranges. We believe this work expands the current capabilities of cell-free circuits by incorporating PSR, which we anticipate will have a wide range of uses within biotechnology.

Water contamination is a growing global concern, creating a need to develop technologies that can detect a range of target compounds at the required thresholds. Here, we address this need by merging biological allosteric transcription factors with DNA coated nanomechanical microcantilevers to detect chemicals in water with digital readout. After proof-of-concept demonstration and optimization to detect tetracycline with the TetR transcription factor, we use the CadC transcription factor to detect Pb ²⁺ and Cd ²⁺ in water at concentrations down to 2 ppb and 1 ppb, respectively, in less than fifteen minutes. A computational model suggests this improvement in sensitivity could be achieved by the DNA coated microcantilever surface changing transcription factor binding properties. Our findings demonstrate a promising new approach for water quality monitoring with fast, highly sensitive, digital readouts.