Engineering a PbrR-Based Biosensor for Cell-Free Detection of Lead at the Legal Limit

Industrialization and failing infrastructure have led to a growing number of irreversible health conditions resulting from chronic lead exposure. While state-of-the-art analytical chemistry methods provide accurate and sensitive detection of lead, they are too slow, expensive, and centralized to be accessible to many. Cell-free biosensors based on allosteric transcription factors (aTFs) can address the need for accessible, on-demand lead detection at the point of use. However, known aTFs, such as PbrR, are unable to detect lead at concentrations regulated by the Environmental Protection Agency (24–72 nM). Here, we develop a rapid cell-free platform for engineering aTF biosensors with improved sensitivity, selectivity, and dynamic range characteristics. We apply this platform to engineer PbrR mutants for a shift in limit of detection from 10 μM to 50 nM lead and demonstrate use of PbrR as a cell-free biosensor. We envision that our workflow could be applied to engineer any aTF.