Antibiotic-Efficient Genetic Cassette for the TEM-1 β-Lactamase That Improves Plasmid Performance

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Antibiotic resistance cassettes, like TEM-1 β-lactamase (Tn3.1), are crucial for genetic engineering but cause issues.
• High β-lactamase levels degrade antibiotics, reducing selective pressure and plasmid retention.

Purpose of the Study:

• To engineer improved TEM-1 β-lactamase genetic cassettes for enhanced plasmid performance.
• To reduce antibiotic consumption and production costs in bacterial cell factories.

Main Methods:

• Developed a minimal-expression TEM-1 β-lactamase cassette (Tn3.1MIN).
• Created a SEVA-compatible version (Ap(pSEVA#1MIN--)).
• Evaluated antibiotic selection efficiency and plasmid retention.

Main Results:

• Tn3.1MIN and Ap(pSEVA#1MIN--) cassettes require 5-fold lower antibiotic concentrations for selection.
• Increased antibiotic half-life (3- to 10-fold) in culture media.
• Improved plasmid retention in bacterial populations.

Conclusions:

• The engineered cassettes offer efficient antibiotic selection and enhanced plasmid stability.
• These cassettes contribute to antibiotic stewardship, reduced costs, and better plasmid performance in biotechnology.