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.

  • Research Article
  • Published:

Man-made cell-like compartments for molecular evolution

Abstract

Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10 7 -fold excess of genes encoding another enzyme.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Cech, T.R. 1986. A model for the RNA-catalyzed replication of RNA. Proc. Natl. Acad. Sci. USA 83: 4360–4363.

    Article  CAS  Google Scholar 

  2. Joyce, G.F. 1989. RNA evolution and the origins of life. Nature 338: 217–224.

    Article  CAS  Google Scholar 

  3. Eigen, M. 1992. Steps towards life. Oxford University Press, Oxford, UK.

    Google Scholar 

  4. Oparin, A.I. 1957. The origin of life on the earth. Oliver and Boyd, London.

    Google Scholar 

  5. Deamer, D.W. 1997. The first living systems: a bioenergetic perspective. Microbiol. Mol. Biol. Rev. 61: 239–261.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Gold, L., Polisky, B., Uhlenbeck, O., and Yarus, M. 1995. Diversity of oligonu-cleotide functions. Annu. Rev. Biochem. 64: 763–797.

    Article  CAS  Google Scholar 

  7. Hager, A.J., Pollard, J.D., and Szostak, J.W. 1996. Ribozymes: aiming at RNA replication and protein synthesis. Chem. Biol. 3: 717–725.

    Article  CAS  Google Scholar 

  8. Smith, G.P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317.

    Article  CAS  Google Scholar 

  9. Clackson, T. and Wells, J.A. 1994. In vitro selection from protein and peptide libraries. Trends Biotechnol. 12: 173–184.

    Article  CAS  Google Scholar 

  10. Lissant, K.J. (ed.). 1984. Emulsions and emulsion technology. Marcel Dekker, New York.

    Google Scholar 

  11. Becher, P. 1957. Emulsions: theory and practice. Reinhold, New York.

    Google Scholar 

  12. Jeltsch, A., Sobotta, T., and Pingoud, A. 1996. Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalyt-ically inactive single mutants. Protein Eng. 9: 413–423.

    Article  CAS  Google Scholar 

  13. Krieg, N.R. and Holt, J. 1984. Bergey's manual of systematic bacteriology. Williams and Wilkins, Baltimore, MD.

    Google Scholar 

  14. Lesley, S.A. 1995. Preparation and use off. coll S-30 extracts. Methods Mol. Biol. 37: 265–278.

    CAS  PubMed  Google Scholar 

  15. Pohl, F.M., Thomae, R., and Karst, A. 1982. Temperature dependence of the activity of DNA-modifying enzymes: endonucleases and DNA ligase. Eur. J. Biochem. 123: 141–152.

    Article  CAS  Google Scholar 

  16. Chen, L., MacMillan, A., and Verdine, G. 1993. Mutational separation of DNA binding from catalysis in a DNA cytosine methyltransferase. J. Am. Chem. Soc. 115: 5318–5319.

    Article  CAS  Google Scholar 

  17. Fastrez, J. 1997. In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes. Mol. Biotechnol. 7: 37–55.

    Article  CAS  Google Scholar 

  18. Mattheakis, L.C., Bhatt, R.R., and Dower, W.J. 1994. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91: 9022–9026.

    Article  CAS  Google Scholar 

  19. Cull, M.G., Miller, J.F., and Schatz, P.J. 1992. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac represser. Proc. Waft Acad. Sci. USA 89: 1865–1869.

    Article  CAS  Google Scholar 

  20. Roberts, R. and Szostak, J. 1997. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 94: 12297–12302.

    Article  CAS  Google Scholar 

  21. Widersten, M. and Mannervik, B. 1995. Glutathione transferases with novel active sites isolated by phage display from a library of random mutants. J. Mol. Biol. 250: 115–122.

    Article  CAS  Google Scholar 

  22. Soumillion, P., Jespers, L., Bouchet, M., Marchand, B.J., Winter, G., and Fastrez, J. 1994. Selection of beta-lactamase on filamentous bacteriophage by catalytic activity. J. Mol. Biol. 237: 415–422.

    Article  CAS  Google Scholar 

  23. Benner, S.A. 1993. Catalysis: design versus selection. Science 261: 1402–1403.

    Article  CAS  Google Scholar 

  24. Stemmer, W.P. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91: 10747–10751.

    Article  CAS  Google Scholar 

  25. Leung, D.W., Chen, E., and Goeddel, D.V. 1989. A method for random mutagen-esis of a defined DNA segment using a modified polymerase chain reaction. Technique 1: 11–15.

    Google Scholar 

  26. Lowman, H.B., Bass, S.H., Simpson, N., and Wells, J.A. 1991. Selecting high-affinity binding proteins by monovalent phage display. Biochemistry 30: 10832–10938.

    Article  CAS  Google Scholar 

  27. Zhao, H., Giver, L, Shao, Z., Affholter, J.A., and Arnold, F.H. 1998. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature Biotechnology 16: 258–261.

    Article  CAS  Google Scholar 

  28. Tawfik, D.S., Green, B.S., Chap, R., Sela, M., and Eshhar, Z. 1993. catELISA: a facile general route to catalytic antibodies. Proc. Natl. Acad. Sci. USA 90: 373–377.

    Article  CAS  Google Scholar 

  29. Eisenthal, R. and Danson, M.J. (eds.). 1993. Enzyme assays—a practical approach. Oxford University Press, Oxford, UK.

    Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., Chiswell, D.J., Hudson, R, and Winter, G. 1991. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19: 4133–4137.

    Article  CAS  Google Scholar 

  32. Wang, A.M., Doyle, M.V., and Mark, D.F. 1989. Quantitation of mRNA by the polymerase chain reaction. Proc. Watt. Acad. Sci. USA 86: 9717–9721.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Dan S. Tawfik or Andrew D. Griffiths.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tawfik, D., Griffiths, A. Man-made cell-like compartments for molecular evolution . Nat Biotechnol 16, 652–656 (1998). https://doi.org/10.1038/nbt0798-652

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0798-652

This article is cited by

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