ORIGINAL ABSTRACT

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.