tRNA-Mediated Plasmid Stabilization for Antibiotic-Free Applications in Escherichia coli

Area of Science:

• Molecular Biology
• Biotechnology
• Industrial Microbiology

Background:

• Antibiotic selection markers are standard for plasmid maintenance in research.
• Industrial scale-up faces challenges with antibiotic use, including cost and antibiotic resistance.
• Antibiotic-free plasmid maintenance methods are highly sought after.

Purpose of the Study:

• To develop and evaluate an antibiotic-free plasmid selection strategy using an essential gene.
• To establish a workflow for efficient plasmid maintenance without antibiotic resistance genes.
• To demonstrate the applicability of the system in industrial fermentation conditions.

Main Methods:

• Utilized the Escherichia coli tryptophan tRNA (trpT) gene as an essential gene-based selection marker.
• Developed a workflow involving a trpT deletion strain and a temperature-sensitive trpT-expressing plasmid.
• Assessed the stability of antibiotic gene-free trpT plasmids with varying copy numbers.

Main Results:

• The trpT-based system demonstrated comparable or superior plasmid stability to antibiotic-based methods.
• The system proved stable under industrially relevant fermentation conditions for biochemical production.
• The small size of trpT facilitates plasmid minimization, enhancing efficiency.

Conclusions:

• The essential gene-based plasmid selection strategy offers a robust and effective antibiotic-free alternative.
• This approach simplifies bioprocesses and contributes to combating the spread of antibiotic resistance.
• The developed system supports the development of more sustainable and cost-effective biotechnological applications.