2024/11/29 by Zachary T. Baumer, Matilda Newton, Lina Löfstrand +3 · 1 voice
Biochemistry, Genetics and Molecular Biology · Environmental Science · #Bacteriophages and microbial interactions #CRISPR and Genetic Engineering #Molecular Biology Techniques and Applications
paper · doi:10.1021/acssynbio.4c00627
openalex publication_date 2024/11/29 · openalex created_date 2025/10/10 · openalex updated_date 2026/06/22
Precise, stringent, post-translational activation of enzymes is essential for many synthetic biology applications. For example, even a few intracellular molecules of unregulated T7 RNA polymerase can result in growth cessation in a bacterium. We sought to mimic the properties of natural enzymes, where activity is regulated ubiquitously by endogenous metabolites. Here we demonstrate that full-length, single subunit T7-derived RNA polymerases (T7 RNAP) can be activated by physiologically relevant concentrations of indoles. We used rational design and directed evolution to identify T7 RNAP variants with minimal transcriptional activity in the absence of indole, and a 29-fold increase in activity with an EC 50 of 344 μM. Indoles control T7-dependent gene expression exogenously, endogenously, and between cells. We also demonstrate indole-dependent bacteriophage viability and propagation in trans. Specificity of different indoles, T7 promoter specificities, and portability to different bacteria are shown. Our l igand a ctivated R NA p olymerases (LARPs) represent a new chemically inducible “stop and go” platform immediately deployable for novel synthetic biology applications, including for modulation of synthetic cocultures.