Expanding allosteric transcription factor-based biosensors through the design and development of a novel estrogen sensor
Embargo Date
2033-01-31
OA Version
Citation
Abstract
Bacterial allosteric transcription factors (aTFs) are core components of molecular biosensors, enabling the selective detection of small molecules with broad applications in diagnostics, environmental monitoring, and synthetic biology. However, their broader potential is restricted by the limited number of aTFs with characterized responsiveness to relevant ligands and by incomplete understanding of their DNA-binding specificity. In this work, a previously uncharacterized aTF from Croceicoccus estronivorus was identified and shown to exhibit estrogen-dependent modulation of DNA binding in vitro, expanding the known functional diversity of allosteric transcription factors. Genome-wide binding assays were performed to define the protein’s binding motif and potential regulatory targets. These data were then used to train a machine learning model capable of predicting protein-DNA binding affinity from sequence alone, which was validated using bio-layer interferometry. Guided by these predictions and measurements, operator variants spanning a range of predicted affinities were designed and tested, revealing that estrogen-induced modulation of TF–DNA binding occurred only at a subset of sites. Using the best performing operator, we established the foundation for the first aTF-based in vitro estrogen optical biosensor. Finally, a high-throughput screening platform was adapted and validated for the discovery of novel aTFs from genomic libraries, addressing the bottleneck of sensing-part discovery. Together, this work establishes a framework for the discovery, characterization, and engineering of aTFs for sensing applications, providing the groundwork for the next generation of biosensor development.
Description
2026