A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range

Area of Science:

• Synthetic biology
• Molecular biology
• Microbial engineering

Background:

• Genetically engineered photoreceptors provide advanced control over gene expression.
• The CcaSR two-component system from Synechocystis PCC 6803, activated by green light, was previously adapted for E. coli (CcaSR v1.0 and v2.0).
• Previous optimizations enhanced dynamic range to ~120-fold and reduced transcriptional leakiness.

Purpose of the Study:

• To develop an improved light-inducible gene expression system.
• To investigate the impact of deleting PAS domains from the CcaS sensor on system performance.
• To create a highly sensitive and dynamic bacterial light sensor.

Main Methods:

• Deletion of two PAS domains from the CcaS histidine kinase component of the CcaSR system.
• Integration of these deletions into the previously optimized CcaSR v2.0 system (creating CcaSR v3.0).
• Characterization of the new system's dynamic range, leakiness, light sensitivity, and response dynamics in E. coli.

Main Results:

• The CcaSR v3.0 system exhibited a dynamic range of nearly 600-fold, a significant improvement.
• Transcriptional leakiness was reduced by approximately fourfold compared to CcaSR v2.0.
• PAS domain deletions did not negatively impact green light sensitivity or response speed.

Conclusions:

• CcaSR v3.0 represents a substantial advancement in engineered bacterial light sensors.
• This enhanced system offers superior performance for precise, quantitative, temporal, and spatial control of gene expression.
• The improved CcaSR v3.0 sensor is expected to find broad utility in fundamental and synthetic biology research.