Fig. 1.

Large-scale sensor isolation to enable data-driven sensor design. (A) The PYR1 sensor isolation pipeline. PYR1 binds to its effector protein HAB1 in response to ABA, its native ligand. This interaction can be measured in yeast growth assays by activating a genetic circuit that rescues uracil auxotrophy. Mutant PYR1 variants that recognize new ligands are identified from receptor library pools using selection experiments (responders grow in the absence of uracil). (B) A high-throughput version of the receptor isolation approach was developed. In this, we plate ~150,000 cells onto media containing a test chemical and select colonies from the wells for retesting and subsequent sequencing. These efforts identified 553 receptors that recognize 181 unique molecules. (C) Chemoinformatic analysis of the data is used to build maps of sequence–ligand interactions. (D) The sequence–ligand maps developed can be used to drive the design of sensors. Libraries of receptors targeted to specific molecule classes are constructed using sequence profiles to design oligonucleotide pools that can be assembled using Golden Gate cloning; subsequent growth-based screens enable one-step isolation of high-affinity sensors.