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Phage-assisted continuous and non-continuous evolution


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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Fig. 1: Overview of PACE.
Fig. 2: Flow diagram of the Procedure.
Fig. 3: Assembly of a PACE apparatus.
Fig. 4: Monitoring the PACE experiment.

Data availability

The authors declare that any data discussed in this protocol are available in the supporting primary research papers.


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We thank K. Esvelt, J. Carlson, B. Dickinson, A. Badran, B. Hubbard, M. Packer, D. Bryson, J. Hu, T. Roth, B. Thuronyi, M. Richter and K. Zhao for their contributions to the development of PACE and T. Blum for helpful discussion. S.M.M. was supported by a National Science Foundation Graduate Research Fellowship. T.W. was supported by a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellowship (F32GM119228). We are grateful for support from US NIH R01 EB027793, U01 AI142756, RM1 HG009490, R35 GM118062, the Howard Hughes Medical Institute and the Bill & Melinda Gates Foundation.

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Authors and Affiliations



S.M.M., T.W. and D.R.L wrote the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

S.M.M., T.W. and D.R.L. have filed patent applications on PACE technologies and PACE-evolved proteins.

Additional information

Peer review information Nature Protocols thanks Mark Isalan, Chang Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Esvelt, K. M., Carlson, J. C. & Liu, D. R. Nature 472, 499−503 (2011):

Carlson, J. C., Badran, A. H., Guggiana-Nilo, D. A. & Liu, D. R. Nat. Chem. Biol. 10, 216−222 (2014):

Badran, A. H. et al. Nature 533, 58−63 (2016):

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Data 1

GenBank-formatted plasmid map depicting ∆gIII selection phage with a rpoz-dSpCas9 insert as the POI. This insert can be swapped for any POI using any standard cloning method.

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Miller, S.M., Wang, T. & Liu, D.R. Phage-assisted continuous and non-continuous evolution. Nat Protoc 15, 4101–4127 (2020).

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