Journal Article

Active learning-guided optimization of cell-free biosensors for lead testing in drinking water

Brown, Dylan M., Chiang, Nicole, Dildine, Garrett, Feng, Siyuan, Gaillard, Jean-François, Karim, Ashty S., Wang, Brenda M., Lucci, Tyler J., Jewett, Michael C., Ekas, Holly M., Lucks, Julius B., Bly, Vanessa, Shukla, Diwakar

2025

Overview

Point-of-use diagnostics based on allosteric transcription factors (aTFs) are promising tools for environmental monitoring and human health. However, biosensors relying on natural aTFs rarely exhibit the sensitivity and selectivity needed for real-world applications, and traditional directed evolution struggles to optimize multiple biosensor properties at once. To overcome these challenges, we develop a multi-objective, machine learning (ML)-guided cell-free gene expression workflow for engineering aTF-based biosensors. Our approach rapidly generates high-quality sequence-to-function data, which we transform into an augmented paired dataset to train an ML model using directional labels that capture how aTF mutations alter performance. We apply our workflow to engineer the aTF PbrR as a point-of-use diagnostic for lead contamination in water. We tune the sensitivity of PbrR to sense at the U.S. Environmental Protection Agency (EPA) action level for lead and modify the selectivity away from zinc, a common metal found in water supplies. Finally, we show that the engineered PbrR functions in freeze-dried cell-free reactions, enabling a diagnostic capable of detecting lead in drinking water down to ~5.7 ppb. Our ML-driven, multi-objective framework powered by directional tokens can generalize to other biosensors and proteins, accelerating the development of synthetic biology tools for biotechnology applications. Allosteric transcription factors (aTFs) are promising tools for environmental and human health monitoring. Here the authors develop a multi-objective, machine learning-guided method to engineer an aTF-based portable diagnostic for environment sensing of lead in drinking water at the legal limit.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/114/2397 / 631/1647/1888 / 631/61/338/552 / 82/47 / Allosteric properties / Biosensing Techniques - methods / Biosensors / Biotechnology / Cell-Free System / Datasets / Design / Directed evolution / Drinking water / Drinking Water - analysis / Drinking Water - chemistry / Engineers / Environmental monitoring / Environmental Monitoring - methods / Environmental protection / Gene expression / Genetic engineering / Humanities and Social Sciences / Humans / Lead / Lead - analysis / Learning algorithms / Ligands / Machine Learning / multidisciplinary / Mutagenesis / Mutation / Optimization / Proteins / Robotics / Science / Science (multidisciplinary) / Sensitivity / Transcription factors / Transcription Factors - genetics / Transcription Factors - metabolism / Water Pollutants, Chemical - analysis / Water pollution / Water supply / Workflow