Directed Evolution in <i>Escherichia coli</i> for Novel Ligand‐Binding Regulators: Evolving a Progesterone‐Responsive Transcription Factor to Bind Cortisol

Allosteric transcription factors (aTFs) regulate gene expression in response to small molecules. Engineering aTFs with new ligand specificities expands their utility in environmental monitoring and diagnostics. Here, we present protocols for improving the responsiveness of an aTF, SRTF1, to its non-native ligand, cortisol, based on the SELIS (Seamless Enrichment of Ligand-Inducible Sensors) methodology. First, an aTF library is generated by randomizing the ligand binding pocket. These libraries are then subjected to a selection process to remove protein variants that have lost the ability to repress transcription. The libraries are then plated onto solid media containing the target ligand. The most fluorescent colonies, signifying the most responsive aTFs, are cultured and assayed for the fold change in fluorescence upon induction with the target ligand. Colonies with the highest fold change are cultured, and the plasmid DNA is purified and sequenced. The gene variants are then subcloned into new expression vectors and assayed in a dose-response assay with the target and non-target ligands to have complete characterization of the sensor. Through one round of these protocols, we improved the response of SRTF1 to cortisol by seven-fold; however, the protocols outlined here are applicable to virtually any protein:effector pair. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Library design Basic Protocol 2: Library construction Basic Protocol 3: Selection and screening Basic Protocol 4: Sequencing and validation of hits Support Protocol 1: Predicting protein:effector structure Support Protocol 2: Design of mutagenesis primers Support Protocol 3: Preparation of chemically competent E. coli for single isolate transformations.