Digitizing the Blue Light-Activated T7 RNA Polymerase System with a <i>tet</i>-Controlled Riboregulator

Optogenetic systems offer precise control over gene expression, but leaky activity in the dark limits their dynamic range and, consequently, their applicability. Here, we enhanced an optogenetic system based on a split T7 RNA polymerase fused to blue-light-inducible Magnets by incorporating a <i>tet</i>-controlled riboregulatory module. This module exploits the photosensitivity of anhydrotetracycline and the designability of synthetic small RNAs to digitize light-controlled gene expression, implementing a repressive action over the translation of a polymerase fragment gene that is relieved with blue light. Our engineered system exhibited 13-fold improvement in dynamic range upon blue light exposure, which even raised to 23-fold improvement when using cells preadapted to chemical induction. As a functional demonstration, we implemented light-controlled antibiotic resistance in bacteria. Such integration of regulatory layers represents a suitable strategy for engineering better circuits for light-based biotechnological applications.