References
Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).
Mastrobattista, E. et al. High-throughput screening of enzyme libraries: in vitro evolution of a β-galactosidase by fluorescence-activated sorting of double emulsions. Chem. Biol. 12, 1291–1300 (2005).
Bernath, K., Magdassi, S. & Tawfik, D.S. Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery. J. Mol. Biol. 345, 1015–1026 (2005).
Griffiths, A.D. & Tawfik, D.S. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J. 22, 24–35 (2003).
Cohen, H.M., Tawfik, D.S. & Griffiths, A.D. Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization. Protein Eng. Des. Sel. 17, 3–11 (2004).
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).
Ghadessy, F.J. & Holliger, P. A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system. Protein Eng. Des. Sel. 17, 201–204 (2004).
Agresti, J.J., Kelly, B.T., Jaschke, A. & Griffiths, A.D. Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc. Natl. Acad. Sci. USA 102, 16170–16175 (2005).
Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).
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).
Doi, N., Kumadaki, S., Oishi, Y., Matsumura, N. & Yanagawa, H. In vitro selection of restriction endonucleases by in vitro compartmentalization. Nucleic Acids Res. 32, e95 (2004).
Sepp, A., Tawfik, D.S. & Griffiths, A.D. Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry. FEBS Lett. 532, 455–458 (2002).
Bertschinger, J. & Neri, D. Covalent DNA display as a novel tool for directed evolution of proteins in vitro. Protein Eng. Des. Sel. 17, 699–707 (2004).
Sepp, A. & Choo, Y. Cell-free selection of zinc finger DNA-binding proteins using in vitro compartmentalization. J. Mol. Biol. 354, 212–219 (2005).
Kemp, B.M. & Smith, D.G. Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci. Int. 154, 53–61 (2005).
Griffiths, A.D. & Tawfik, D.S. Man-made enzymes-from design to in vitro compartmentalisation. Curr. Opin. Biotechnol. 11, 338–353 (2000).
Umbanhowar, P.B., Prasad, V. & Weitz, D.A. Monodisperse emulsions generated via drop break off in a coflowing steam. Langmuir 16, 347–351 (2000).
Thorsen, T., Roberts, R.W., Arnold, F.H. & Quake, S.R. Dynamic pattern formation in a vesicle-generating microfluidic device. Phys. Rev. Lett. 86, 4163–4166 (2001).
Anna, S.L., Bontoux, N. & Stone, H.A. Formation of dispersions using “flow focusing” in microchannels. Appl. Physics Lett. 82, 364–366 (2003).
Utada, A.S. et al. Monodisperse double emulsions generated from a microcapillary device. Science 308, 537–541 (2005).
Lee, Y.F., Tawfik, D.S. & Griffiths, A.D. Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation. Nucleic Acids Res. 30, 4937-4944 (2002).
Ghadessy, F.J. et al. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Nat. Biotechnol. 22, 755–759 (2004).
Yonezawa, M., Doi, N., Kawahashi, Y., Higashinakagawa, T. & Yanagawa, H. DNA display for in vitro selection of diverse peptide libraries. Nucleic Acids Res. 31, e118 (2003).
Yonezawa, M., Doi, N., Higashinakagawa, T. & Yanagawa, H. DNA display of biologically active proteins for in vitro protein selection. J. Biochem. (Tokyo) 135, 285–288 (2004).
Levy, M., Griswold, K.E. & Ellington, A.D. Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization. RNA 11, 1555–1562 (2005).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The Medical Research Council (MRC) and the Weizmann Institute have filed patent applications, which include some of the ideas described in this manuscript. Should these organizations receive revenues from licensing these patents, the authors may be entitled to receive payments through the organizations’ Rewards to Inventors schemes.
Supplementary information
Rights and permissions
About this article
Cite this article
Miller, O., Bernath, K., Agresti, J. et al. Directed evolution by in vitro compartmentalization. Nat Methods 3, 561–570 (2006). https://doi.org/10.1038/nmeth897
Issue Date:
DOI: https://doi.org/10.1038/nmeth897
- Springer Nature America, Inc.
This article is cited by
-
Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
Scientific Reports (2018)
-
RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
Nature Communications (2017)
-
Droplet-based digital PCR system for detection of single-cell level of foodborne pathogens
BioChip Journal (2017)
-
Cell-free systems in the new age of synthetic biology
Frontiers of Chemical Science and Engineering (2017)