Phage-assisted continuous and non-continuous evolution.

Nature Protocols
Shannon M MillerDavid R Liu

Abstract

Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-continuous evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a continuous flow apparatus and the performance and analysis of evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.

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Citations

Mar 31, 2021·Current Opinion in Chemical Biology·Gordon Rix, Chang C Liu
Jul 29, 2021·ACS Synthetic Biology·Jeffrey A DeweyBryan C Dickinson
Oct 15, 2021·Nature Communications·Mary S MorrisonDavid R Liu

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Datasets Mentioned

BETA
AB1396

Methods Mentioned

BETA
deamination
two-hybrid
PCR

Software Mentioned

PACE
Addgene
PANCE
OrthoRep
PACEmid

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