A destabilized bacterial luciferase for dynamic gene expression studies.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• Genetic regulatory elements fused with reporter genes are crucial for monitoring gene expression and synthetic gene circuits.
• Long reporter protein half-lives limit real-time transcription monitoring and can degrade synthetic circuit performance.

Purpose of the Study:

• To develop short half-life variants of bacterial luciferase (LuxA and LuxB) for improved real-time gene expression monitoring.
• To apply C-terminal tagging for protein half-life regulation in a multi-subunit enzyme complex.

Main Methods:

• Constructed short half-life variants of LuxA and LuxB from Photorhabdus luminescens in Escherichia coli.
• Incorporated an 11-amino acid carboxy-terminal tag recognized by endogenous tail-specific proteases.
• Altered terminal residues of the C-terminal tag to modulate protein half-life.

Main Results:

• The C-terminal tag significantly affected the functional half-life of the holoenzyme when fused to LuxA or both LuxA and LuxB.
• Modification of LuxB alone did not substantially alter protein half-life.
• Altering the tag's terminal residues generated variants with intermediate half-lives, similar to GFP.

Conclusions:

• C-terminal tagging effectively regulates protein half-life for bacterial luciferase reporter genes.
• This approach enhances the utility of luciferase reporters for dynamic gene expression monitoring in synthetic biology.
• This is the first application of C-terminal tagging to regulate the half-life of an enzyme or its subunits.