A Plasmid-Based Fluorescence Reporter System for Monitoring Oxidative Damage in E. coli

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Quantifying intracellular oxidative damage from reactive oxygen species (ROS) is crucial in biological research.
• Existing methods often rely on exogenous, oxygen-sensitive substrates requiring cell penetration and spectroscopic monitoring.
• A need exists for non-invasive strategies to assess ROS-induced damage within living cells.

Purpose of the Study:

• To design and validate a novel, non-invasive analytical strategy for measuring ROS-induced damage in living microbial cells.
• To leverage the native redox sensor system of *E. coli* for detecting oxidative stress.
• To develop a cost-effective and simple method for analyzing enzyme-level oxidative damage.

Main Methods:

• Engineered a plasmid-based biosensor in *E. coli* utilizing the oxygen-sensitive transcriptional repressor IscR.
• IscR controls the expression of an in vivo fluorescent marker, allowing for real-time monitoring of oxidative stress.
• Validated the sensor's response to hydrogen peroxide (H₂O₂) and low cultivation temperatures, comparing results with fluorescence microscopy and a commercial probe (CellROX).

Main Results:

• The developed plasmid-based sensor quantitatively responded to induced oxidative stress.
• The system demonstrated specificity comparable to the commercial chemical probe CellROX.
• The strategy successfully detected enzyme-level oxidative damage in *E. coli* without the need for chemical probes.

Conclusions:

• A novel, non-invasive, and inexpensive fluorescent reporter system was established for quantifying oxidative damage in *E. coli*.
• This method utilizes *E. coli*'s native redox sensing capabilities, avoiding reliance on external chemical probes.
• The developed strategy offers a simple and effective tool for analyzing oxidative stress in microbial systems.