Preventing Multimer Formation in Commonly Used Synthetic Biology Plasmids

Area of Science:

• Molecular Biology
• Synthetic Biology
• Microbiology

Background:

• Plasmids are vital tools in research and biotechnology.
• Maintaining plasmid integrity, specifically preventing multimer formation, is crucial for optimizing synthetic circuits.
• Plasmid multimers, arising from failed dimer resolution, can impact circuit function, particularly with DNA-editing enzymes.

Purpose of the Study:

• To investigate the occurrence of plasmid multimers in common laboratory strains of Escherichia coli.
• To identify factors influencing multimer formation.
• To develop strategies for mitigating multimer production in plasmid-based systems.

Main Methods:

• Surveyed four common plasmid backbones in cloning (JM109) and wild-type (MG1655) Escherichia coli strains.
• Assessed multimer formation in relation to plasmid copy number and culture passaging.
• Created and tested an MG1655 ΔrecA single-locus knockout strain for its ability to prevent multimer production.

Main Results:

• Plasmid multimers were observed significantly only in the MG1655 strain.
• Multimer fraction increased with higher plasmid copy number and extended culture passaging in MG1655.
• Transformed multimers into JM109 resulted in strains lacking singlet plasmids.
• The MG1655 ΔrecA knockout strain effectively prevented multimer formation.

Conclusions:

• Plasmid multimerization is strain-dependent in Escherichia coli, occurring notably in wild-type MG1655.
• Factors like plasmid copy number and passaging influence multimer accumulation.
• Genetic modification, such as creating a ΔrecA knockout, can eliminate multimer production, enhancing plasmid-based synthetic circuit reliability.