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Phage-assisted continuous and non-continuous evolution.

Shannon M Miller1,2,3, Tina Wang1,2,3,4, David R Liu5,6,7

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Directed evolution in the lab creates new biomolecules. Phage-assisted continuous evolution (PACE) accelerates this process, enabling over 100 evolution rounds in just two weeks.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Directed evolution mimics Darwinian principles in vitro to engineer biomolecules.
  • Continuous evolution methods streamline directed evolution, reducing researcher intervention.
  • Phage-assisted continuous evolution (PACE) leverages bacteriophage life cycles for rapid, in vivo evolution in bacteria.

Purpose of the Study:

  • To provide a detailed protocol for protein evolution using PACE and PANCE.
  • To enable rapid, high-throughput generation of tailored biomolecules.
  • To demonstrate the efficiency of continuous evolution for accelerated Darwinian selection.

Main Methods:

  • Utilizing PACE to perform continuous directed evolution in bacteria.
  • Implementing phage-assisted non-continuous evolution (PANCE) protocols.
  • Detailed instructions for preparing selection phage and host cells.
  • Assembly and operation of a continuous flow apparatus for evolution experiments.

Main Results:

  • PACE enables over 100 rounds of evolution (mutation, selection, replication) in a single experiment.
  • Evolution experiments can be completed in as little as 2 weeks.
  • Facilitates directed evolution on significantly faster timescales than conventional methods.

Conclusions:

  • PACE and PANCE are powerful, efficient protocols for protein evolution.
  • Continuous evolution significantly accelerates the generation of novel biomolecules.
  • This protocol empowers researchers to rapidly tailor biomolecular properties.