Compartmentalized partnered replication for the directed evolution of genetic parts and circuits


Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype–genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take 3–5 d, whereas a whole directed evolution can be performed in 3–10 rounds, depending on selection efficiency.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: General CPR concept.
Figure 2: Overview and time line of experiments.
Figure 3: Schematic of the two Recovery strategies.
Figure 4: Anticipated results.


  1. 1

    Romero, P.A. & Arnold, F.H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

  2. 2

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

  3. 3

    Gillam, E.M.J., Copp, J.N. & Ackerley, D.F. Directed Evolution Library Creation: Methods and Protocols 2nd edn (Humana Press, 2014).

  4. 4

    Tee, K.L. & Wong, T.S. Polishing the craft of genetic diversity creation in directed evolution. Biotechnol. Adv. 31, 1707–1721 (2013).

  5. 5

    Boder, E.T. & Wittrup, K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

  6. 6

    Bessette, P.H., Rice, J.J. & Daugherty, P.S. Rapid isolation of high-affinity protein binding peptides using bacterial display. Protein Eng. Des. Sel. 17, 731–739 (2004).

  7. 7

    McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

  8. 8

    Hanes, J. & Pluckthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942 (1997).

  9. 9

    Wilson, D.S., Keefe, A.D. & Szostak, J.W. The use of mRNA display to select high-affinity protein-binding peptides. Proc. Natl. Acad. Sci. USA 98, 3750–3755 (2001).

  10. 10

    Leemhuis, H., Stein, V., Griffiths, A.D. & Hollfelder, F. New genotype-phenotype linkages for directed evolution of functional proteins. Curr. Opin. Struct. Biol. 15, 472–478 (2005).

  11. 11

    Amstutz, P. et al. In vitro selection for catalytic activity with ribosome display. J. Am. Chem. Soc. 124, 9396–9403 (2002).

  12. 12

    Seelig, B. & Szostak, J.W. Selection and evolution of enzymes from a partially randomized non-catalytic scaffold. Nature 448, 828–831 (2007).

  13. 13

    Cesaro-Tadic, S. et al. Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library. Nat. Biotechnol. 21, 679–685 (2003).

  14. 14

    Palmer, A.C. & Kishony, R. Understanding, predicting and manipulating the genotypic evolution of antibiotic resistance. Nat. Rev. Genet. 14, 243–248 (2013).

  15. 15

    Dietrich, J.A., McKee, A.E. & Keasling, J.D. High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu. Rev. Biochem. 79, 563–590 (2010).

  16. 16

    Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).

  17. 17

    Ghadessy, F.J., Ong, J.L. & Holliger, P. Directed evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci. USA 98, 4552–4557 (2001).

  18. 18

    Ghadessy, F.J. & Holliger, P. Compartmentalized self-replication: a novel method for the directed evolution of polymerases and other enzymes. Methods Mol. Biol. 352, 237–248 (2007).

  19. 19

    Loakes, D., Gallego, J., Pinheiro, V.B., Kool, E.T. & Holliger, P. Evolving a polymerase for hydrophobic base analogues. J. Am. Chem. Soc. 131, 14827–14837 (2009).

  20. 20

    Ramsay, N. et al. CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase. J. Am. Chem. Soc. 132, 5096–5104 (2010).

  21. 21

    d'Abbadie, M. et al. Molecular breeding of polymerases for amplification of ancient DNA. Nat. Biotechnol. 25, 939–943 (2007).

  22. 22

    Ellefson, J.W. et al. Synthetic evolutionary origin of a proofreading reverse transcriptase. Science 352, 1590–1593 (2016).

  23. 23

    Aharoni, A., Amitai, G., Bernath, K., Magdassi, S. & Tawfik, D.S. High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. Chem. Biol. 12, 1281–1289 (2005).

  24. 24

    Mastrobattista, E. et al. High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions. Chem. Biol. 12, 1291–1300 (2005).

  25. 25

    Davies, D. Cell separations by flow cytometry. Methods Mol. Biol. 878, 185–199 (2012).

  26. 26

    Colin, P.Y., Zinchenko, A. & Hollfelder, F. Enzyme engineering in biomimetic compartments. Curr. Opin. Struct. Biol. 33, 42–51 (2015).

  27. 27

    Meyer, A.J., Ellefson, J.W. & Ellington, A.D. Directed evolution of a panel of orthogonal T7 RNA polymerase variants for in vivo or in vitro synthetic circuitry. ACS Synth. Biol. 4, 1070–1076 (2015).

  28. 28

    Ellefson, J.W. et al. Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat. Biotechnol. 32, 97–101 (2014).

  29. 29

    Maranhao, A.C. & Ellington, A.D. Evolving orthogonal suppressor tRNAs to incorporate modified amino acids. ACS Synth. Biol. 6, 108–119 (2017).

  30. 30

    Yang, G. & Withers, S.G. Ultrahigh-throughput FACS-based screening for directed enzyme evolution. Chembiochem 10, 2704–2715 (2009).

  31. 31

    van Rossum, T., Kengen, S.W. & van der Oost, J. Reporter-based screening and selection of enzymes. FEBS J. 280, 2979–2996 (2013).

  32. 32

    Leung, D.W., Chen, E. & Goeddel, D.V. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1, 11–15 (1989).

  33. 33

    Cadwell, R.C. & Joyce, G.F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).

  34. 34

    Hutchison, C.A. III et al. Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253, 6551–6560 (1978).

  35. 35

    Green, M.R., Sambrook, J. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn (Cold Spring Harbor Laboratory Press, 2012).

  36. 36

    Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  37. 37

    Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647 (2008).

  38. 38

    Miller, E.M. & Nickoloff, J.A. Escherichia coli electrotransformation. Methods Mol. Biol. 47, 105–113 (1995).

  39. 39

    Sambrook, J. & Russell, D.W. Transformation of E. coli by electroporation. CSH Protoc. (2006).

  40. 40

    Diehl, F. et al. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat. Methods 3, 551–559 (2006).

  41. 41

    Williams, R. et al. Amplification of complex gene libraries by emulsion PCR. Nat. Methods 3, 545–550 (2006).

  42. 42

    Nakano, M. et al. Single-molecule PCR using water-in-oil emulsion. J. Biotechnol. 102, 117–124 (2003).

  43. 43

    McDonald, J.C. et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21, 27–40 (2000).

  44. 44

    Mazutis, L. et al. Single-cell analysis and sorting using droplet-based microfluidics. Nat. Protoc. 8, 870–891 (2013).

  45. 45

    Schutze, T. et al. A streamlined protocol for emulsion polymerase chain reaction and subsequent purification. Anal. Biochem. 410, 155–157 (2011).

  46. 46

    Kelly, J.R. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J. Biol. Eng. 3, 4 (2009).

  47. 47

    Davis, J.H., Rubin, A.J. & Sauer, R.T. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39, 1131–1141 (2011).

  48. 48

    Bonde, M.T. et al. Predictable tuning of protein expression in bacteria. Nat. Methods 13, 233–236 (2016).

  49. 49

    Mikula, R.J. Emulsion characterization. Adv. Chem. Ser. 231, 79–129 (1992).

Download references


This work was supported by the Welch Foundation (F-1654 to A.D.E.), the DOD Air Force Research Laboratory (FA9550-14-1-0089), Firebird Biomolecular Sciences (1R41GM119434-01A1), and the John Templeton Foundation (54466). The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation.

Author information

Z.A. and A.D.E. wrote the manuscript. Z.A., J.W.E., J.D.G., E.W. and A.D.E. contributed technical detail to the protocol, and read, edited, and approved the final manuscript.

Correspondence to Andrew D Ellington.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Basic plasmid schemes.

pACYC-Taq was modified to make pACYC-Taq.1Amb, pACYC-GFP, pACYC-GFP1Amb, and pACYC-GFPM2 by cloning the appropriate coding DNA sequence (CDS) in the place of the Taq DNAP CDS by isothermal (Gibson) assembly. Promoter mutations and amber mutations were made in pACYC plasmids by isothermal assembly with mutagenic primers. pRST.11B-AS3.4 encodes suppressor tRNA that was previously rationally engineered from WT yeast suppressor tRNA for improved amber suppression. This plasmid was used as a parental plasmid for construction of tRNA synthetase and tRNA libraries for CPR selections. Reprinted by permission from Macmillan Publishers Ltd: Nat Biotechnol, copyright 2014. (Ellefson et al. Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat Biotechnol 32, 97-101, doi:10.1038/nbt.2714 (2014).).

Supplementary Figure 2 Assembly of the full-length T7 RNAP gene by overlap extension PCR.

Fragments are not drawn to scale.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1 and 2, Supplementary Note and Supplementary Table 1. (PDF 508 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Abil, Z., Ellefson, J., Gollihar, J. et al. Compartmentalized partnered replication for the directed evolution of genetic parts and circuits. Nat Protoc 12, 2493–2512 (2017).

Download citation

Further reading


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.