Quantifying plasmid copy number to investigate plasmid dosage effects associated with directed protein evolution

Area of Science:

• Molecular Biology
• Biochemistry
• Evolutionary Biology

Background:

• Directed protein evolution aims to engineer proteins under specific laboratory conditions.
• ColE1 plasmids are commonly used for in vivo library generation in protein evolution studies.
• Mutations outside target genes can influence evolutionary outcomes.

Purpose of the Study:

• To investigate the impact of non-coding mutations on directed protein evolution.
• To understand the role of plasmid origin of replication (ori) mutations in protein evolution.
• To present methods for quantifying plasmid copy number and its effect on protein evolution.

Main Methods:

• Utilizing directed protein evolution with target genes on ColE1 plasmids.
• Introducing random mutations not restricted to coding sequences.
• Quantifying plasmid copy number using established laboratory methods.

Main Results:

• Directed evolution strongly selects for mutations in the plasmid origin of replication (ori).
• Selected ori mutations lead to varied plasmid copy numbers.
• This selection occurs even when evolving novel biochemical activities.
• Plasmid copy number variations suggest a complex interaction with coding mutations.

Conclusions:

• Plasmid origin of replication (ori) mutations are a significant factor in directed protein evolution.
• Plasmid copy number can be a critical evolutionary parameter, fine-tuning protein activity levels.
• The interplay between ori and coding mutations is more complex than simple expression level changes.