Article | Published:

Biocontainment of genetically modified organisms by synthetic protein design

Nature volume 518, pages 5560 (05 February 2015) | Download Citation

  • A Corrigendum to this article was published on 23 September 2015


Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient because they impose evolutionary pressure on the organism to eject the safeguard by spontaneous mutagenesis or horizontal gene transfer, or because they can be circumvented by environmentally available compounds. Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code (Escherichia coli strain C321.ΔA) to confer metabolic dependence on non-standard amino acids for survival. The resulting GMOs cannot metabolically bypass their biocontainment mechanisms using known environmental compounds, and they exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfer. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by a reliance on synthetic metabolites.

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Protein Data Bank

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Atomic coordinates and structure factors for the reported crystal structure have been deposited in the Protein Data Bank under accession number 4OUD.


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We thank D. Renfrew for help with NSAA modelling in Rosetta, D. Goodman and R. Chari for sequence analysis assistance, M. Napolitano for advice on Lon-mediated escape assays, J. Teramoto and B. Wanner for the pJTE2 jumpstart plasmid, and F. Isaacs for manuscript comments. D.J.M. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. M.J.L. was supported by a US Department of Defense National Defense Science and Engineering Graduate Fellowship. M.T.M. was supported by a Doctoral Study Award from the Canadian Institutes of Health Research. The research was supported by Department of Energy Grant DE-FG02-02ER63445.

Author information

Author notes

    • Daniel J. Mandell
    •  & Marc J. Lajoie

    These authors contributed equally to this work.


  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Daniel J. Mandell
    • , Marc J. Lajoie
    • , Michael T. Mee
    • , Gleb Kuznetsov
    • , Julie E. Norville
    • , Christopher J. Gregg
    •  & George M. Church
  2. Program in Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Marc J. Lajoie
  3. Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA

    • Michael T. Mee
  4. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA

    • Ryo Takeuchi
    •  & Barry L. Stoddard
  5. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA

    • George M. Church


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D.J.M., M.J.L., M.T.M. and G.M.C. conceived the project and designed the study, with D.J.M. as computational lead and M.J.L. as experimental lead. D.J.M. computationally designed synthetic auxotrophs, performed strain engineering, characterized escape frequencies and fitness of synthetic auxotrophs, performed western blot analyses and prepared samples for mass spectrometry and X-ray crystallography. M.J.L. performed strain engineering, performed site-saturation mutagenesis at UAG positions, performed whole-genome sequencing of escapees, validated escape mechanisms and assessed HGT by conjugation. M.T.M. measured escape frequencies and fitness of natural metabolic auxotrophs, performed competition assays and assessed HGT by conjugation. R.T. and B.L.S. crystallized tyrS.d7 and determined the X-ray structure. G.K. analysed whole-genome sequencing data of escapees. J.E.N. and C.J.G. developed the tdk selection protocol. D.J.M., M.J.L. and M.T.M. wrote the paper.

Competing interests

Harvard has filed a provisional patent application. G.M.C. is a founder of Enevolv Inc. and Gen9bio. Other potentially relevant financial interests are listed at

Corresponding author

Correspondence to George M. Church.

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