Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Phage-assisted continuous and non-continuous evolution

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015).

    CAS  Article  Google Scholar 

  2. 2.

    Badran, A. H. & Liu, D. R. In vivo continuous directed evolution. Curr. Opin. Chem. Biol. 24, 1–10 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Mills, D. R., Peterson, R. L. & Spiegelman, S. An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc. Natl Acad. Sci. USA 58, 217–224 (1967).

    CAS  Article  Google Scholar 

  4. 4.

    Wright, M. C. & Joyce, G. F. Continuous in vitro evolution of catalytic function. Science 276, 614–617 (1997).

    CAS  Article  Google Scholar 

  5. 5.

    Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011).

    CAS  Article  Google Scholar 

  6. 6.

    Berman, C. M. et al. An adaptable platform for directed evolution in human cells. J. Am. Chem. Soc. 140, 18093–18103 (2018).

    CAS  Article  Google Scholar 

  7. 7.

    Badran, A. H. et al. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature 533, 58–63 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Badran, A. H. & Liu, D. R. Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat. Commun. 6, 8425 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Barbas, C. F. Phage Display: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001).

  10. 10.

    Carlson, J. C., Badran, A. H., Guggiana-Nilo, D. A. & Liu, D. R. Negative selection and stringency modulation in phage-assisted continuous evolution. Nat. Chem. Biol. 10, 216–222 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Bennett, N. J. & Rakonjac, J. Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII. J. Mol. Biol. 356, 266–273 (2006).

    CAS  Article  Google Scholar 

  12. 12.

    Hubbard, B. P. et al. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nat. Methods 12, 939–942 (2015).

    CAS  Article  Google Scholar 

  13. 13.

    Leconte, A. M. et al. A population-based experimental model for protein evolution: effects of mutation rate and selection stringency on evolutionary outcomes. Biochemistry 52, 1490–1499 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Packer, M. S., Rees, H. A. & Liu, D. R. Phage-assisted continuous evolution of proteases with altered substrate specificity. Nat. Commun. 8, 956 (2017).

    Article  Google Scholar 

  15. 15.

    Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).

    CAS  Article  Google Scholar 

  16. 16.

    Wang, T., Badran, A. H., Huang, T. P. & Liu, D. R. Continuous directed evolution of proteins with improved soluble expression. Nat. Chem. Biol. 14, 972–980 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Bryson, D. I. et al. Continuous directed evolution of aminoacyl-tRNA synthetases. Nat. Chem. Biol. 13, 1253–1260 (2017).

    CAS  Article  Google Scholar 

  18. 18.

    Roth, T. B., Woolston, B. M., Stephanopoulos, G. & Liu, D. R. Phage-assisted evolution of Bacillus methanolicus methanol dehydrogenase 2. ACS Synth. Biol. 8, 796–806 (2019).

    CAS  Article  Google Scholar 

  19. 19.

    Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    CAS  Article  Google Scholar 

  20. 20.

    Suzuki, T. et al. Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nat. Chem. Biol. 13, 1261–1266 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).

    CAS  Article  Google Scholar 

  22. 22.

    Dickinson, B. C., Leconte, A. M., Allen, B., Esvelt, K. M. & Liu, D. R. Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution. Proc. Natl Acad. Sci. USA 110, 9007–9012 (2013).

    CAS  Article  Google Scholar 

  23. 23.

    Brodel, A. K., Jaramillo, A. & Isalan, M. Engineering orthogonal dual transcription factors for multi-input synthetic promoters. Nat. Commun. 7, 13858 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Pu, J., Zinkus-Boltz, J. & Dickinson, B. C. Evolution of a split RNA polymerase as a versatile biosensor platform. Nat. Chem. Biol. 13, 432–438 (2017).

    CAS  Article  Google Scholar 

  25. 25.

    Zinkus-Boltz, J., DeValk, C. & Dickinson, B. C. A phage-assisted continuous selection approach for deep mutational scanning of protein–protein interactions. ACS Chem. Biol. 14, 2757–2767 (2019).

    CAS  Article  Google Scholar 

  26. 26.

    Pu, J., Disare, M. & Dickinson, B. C. Evolution of C-terminal modification tolerance in full-length and split T7 RNA polymerase biosensors. Chembiochem 20, 1547–1553 (2019).

    CAS  Article  Google Scholar 

  27. 27.

    Dickinson, B. C., Packer, M. S., Badran, A. H. & Liu, D. R. A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations. Nat. Commun. 5, 5352 (2014).

    CAS  Article  Google Scholar 

  28. 28.

    Richter, M. F. Z. et al. Phage-assisted evolution of an adenine base editor with enhanced Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).

    CAS  Article  Google Scholar 

  29. 29.

    Brodel, A. K., Jaramillo, A. & Isalan, M. Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication. Nat. Protoc. 12, 1830–1843 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Javanpour, A. A. & Liu, C. C. Genetic compatibility and extensibility of orthogonal replication. ACS Synth. Biol. 8, 1249–1256 (2019).

    CAS  Article  Google Scholar 

  31. 31.

    Ravikumar, A., Arzumanyan, G. A., Obadi, M. K. A., Javanpour, A. A. & Liu, C. C. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. Cell 175, 1946–1957 (2018).

    CAS  Article  Google Scholar 

  32. 32.

    Zhong, Z., Ravikumar, A. & Liu, C. C. Tunable expression systems for orthogonal DNA replication. ACS Synth. Biol. 7, 2930–2934 (2018).

    CAS  Article  Google Scholar 

  33. 33.

    Zhong, Z. et al. Automated continuous evolution of proteins in vivo. ACS Synth. Biol. 9, 1270–1276 (2020).

    CAS  Article  Google Scholar 

  34. 34.

    English, J. G. et al. VEGAS as a platform for facile directed evolution in mammalian cells. Cell 178, 1030 (2019).

    CAS  Article  Google Scholar 

  35. 35.

    Barbas, C. F. 3rd Recent advances in phage display. Curr. Opin. Biotechnol. 4, 526–530 (1993).

    CAS  Article  Google Scholar 

  36. 36.

    Ringquist, S. et al. Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol. Microbiol. 6, 1219–1229 (1992).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

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

Corresponding author

Correspondence to David R. Liu.

Ethics declarations

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): https://doi.org/10.1038/nature09929

Carlson, J. C., Badran, A. H., Guggiana-Nilo, D. A. & Liu, D. R. Nat. Chem. Biol. 10, 216−222 (2014): https://doi.org/10.1038/nchembio.1453

Badran, A. H. et al. Nature 533, 58−63 (2016): https://doi.org/10.1038/nature17938

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Miller, S.M., Wang, T. & Liu, D.R. Phage-assisted continuous and non-continuous evolution. Nat Protoc 15, 4101–4127 (2020). https://doi.org/10.1038/s41596-020-00410-3

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing