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Updated: May 14, 2026
11:36
A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
A population-based experimental model for protein evolution: effects of mutation rate and selection stringency on
Aaron M Leconte1, Bryan C Dickinson, David D Yang
1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.
Biochemistry
|January 31, 2013
Summary
Protein evolution experiments using phage-assisted continuous evolution (PACE) reveal how mutation rates and selection stringency impact outcomes. This method helps generate novel molecules and understand evolutionary determinants.
Area of Science:
- Biochemistry
- Molecular Biology
- Evolutionary Biology
Background:
- Protein evolution is crucial for organismal development and laboratory-based molecule generation.
- Limited experimental data exists on how key parameters affect protein evolution over extended periods or multiple trials.
Purpose of the Study:
- To experimentally investigate the influence of mutation rate and selection stringency on protein evolution outcomes.
- To characterize convergent genetic solutions during long-term protein evolution using phage-assisted continuous evolution (PACE).
Main Methods:
- Utilized phage-assisted continuous evolution (PACE) for high-throughput, long-term protein evolution experiments.
- Evolved T7 RNA polymerase to recognize the T3 promoter DNA sequence under varied mutation rates and selection stringencies.
- Employed high-throughput sequencing to identify convergent mutations and genetic pathways.
Main Results:
- Demonstrated significant increases in RNA polymerase activity on the target promoter across all tested conditions.
- Identified reproducible evolutionary outcomes influenced by specific combinations of mutation rate and selection stringency.
- Quantitatively defined convergent genetic solutions, including mutational signatures and non-signature mutations.
Conclusions:
- PACE is a valuable platform for studying protein evolution and generating novel molecules.
- Mutation rate and selection stringency are key determinants shaping evolutionary trajectories and outcomes.
- Findings illuminate evolutionary principles and inform the design of future protein engineering experiments.
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