Design of growth-coupled measurements of protease function

This article outlines a proposal for developing a high-throughput measurement platform for protease function using a gene circuit that links protease activity to bacterial cell growth. The system is designed to measure the activity of proteases, particularly focusing on the Tobacco Etch Virus (TEV) protease as an initial target. The circuit relies on the cleavage of lysozyme from a T7 RNA polymerase, which then enables gene expression and antibiotic resistance. The proposal details a two-stage approach for assay development, including tuning the system's dynamic range and establishing normalization and calibration controls. The project aims to collect data on thousands of protease sequence variants against multiple substrates, with potential applications in understanding protease specificity and developing therapeutic interventions. The authors also propose expanding the study to include structural homologs of TEV protease and non-canonical substrates, which could have implications for drug development and understanding protease evolution.