Plasmid selection in Escherichia coli using an endogenous essential gene marker

Area of Science:

• Molecular Biology
• Microbiology
• Biotechnology

Background:

• Antibiotic resistance genes are common plasmid selection tools but risk spreading resistance.
• Existing non-antibiotic systems have limitations like mutant strains or high costs.
• Over-expressing a host essential gene with a specific inhibitor is an unexplored selection concept.

Purpose of the Study:

• To investigate essential gene over-expression for plasmid selection in E. coli.
• To develop a non-antibiotic based plasmid selection system.

Main Methods:

• Utilized the essential gene fabI as a plasmid-borne marker.
• Employed the biocide triclosan as the selective agent.
• Developed a new cloning vector, pFab, for E. coli.

Main Results:

• The pFab vector enabled triclosan-mediated selection at 1 microM.
• pFab demonstrated superior performance over pUC19-ampicillin in cell growth, plasmid stability, and yield.
• A synergistic effect was observed where pFab and triclosan enhanced growth and plasmid production.

Conclusions:

• The fabI-triclosan system offers a viable alternative to antibiotic resistance genes for plasmid selection.
• This method avoids the use of conventional antibiotics and reduces the risk of spreading antibiotic resistance.
• Essential gene over-expression provides a novel strategy for microbial selection.