# Digitizing the Blue Light-Activated T7 RNA Polymerase System with a tet-Controlled Riboregulator

## TL;DR

This paper improves an optogenetic system by adding a riboregulator, allowing better control of gene expression with light.

## Contribution

The novel integration of a tet-controlled riboregulator with a blue-light-inducible T7 RNA polymerase enhances dynamic range and control.

## Key findings

- The system showed a 13-fold improvement in dynamic range under blue light.
- Pre-adapted cells achieved a 23-fold improvement in dynamic range.
- Light-controlled antibiotic resistance was successfully demonstrated in bacteria.

## Abstract

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 tet-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.