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Negative selection and stringency modulation in phage-assisted continuous evolution

Nature Chemical Biology volume 10, pages 216–222 (2014)Cite this article

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Abstract

Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to substantially accelerate laboratory evolution experiments. In this work, we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule–controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counterselection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates while maintaining wild type–like levels of activity.

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Figure 1: Overview of PACE with real-time monitoring.
Figure 2: Drift cassette enables ATc-dependent, activity-independent phage propagation.
Figure 3: Dominant-negative pIII-neg is a potent inhibitor of phage propagation.
Figure 4: Continuous evolution of T3-specific RNAP variants.
Figure 5: Analysis of evolved T7 RNAP mutations that confer PT3 specificity.

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Acknowledgements

The authors thank K. Esvelt, D. Thompson, B. Dorr and K. Davis for helpful discussions. This research was supported by DARPA HR0011-11-2-0003, DARPA N66001-12-C-4207 and the Howard Hughes Medical Institute. J.C.C. was supported by the Harvard Chemical Biology Graduate Program. A.H.B. was supported by a National Science Foundation Graduate Research Fellowship. D.A.G.-N. was supported by the Harvard Biophysics Graduate Program.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Jacob C Carlson, Ahmed H Badran & David R Liu

  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Jacob C Carlson, Ahmed H Badran & David R Liu

  3. Harvard Medical School, Boston, Massachusetts, USA

    Drago A Guggiana-Nilo

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  1. Jacob C Carlson
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  4. David R Liu
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Contributions

J.C.C. designed the research, prepared materials and performed experiments. A.H.B. prepared materials and performed experiments. D.A.G.-N. designed and constructed the real-time luminescence monitor. D.R.L. designed and supervised the research. All of the authors contributed to the manuscript.

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Correspondence to David R Liu.

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The authors have filed a provisional patent application on PACE and related improvements.

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Supplementary Results, Supplementary Figures 1–5 and Supplementary Table 1. (PDF 5132 kb)

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Carlson, J., Badran, A., Guggiana-Nilo, D. et al. Negative selection and stringency modulation in phage-assisted continuous evolution. Nat Chem Biol 10, 216–222 (2014). https://doi.org/10.1038/nchembio.1453

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