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Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells

View ORCID ProfileAditya M. Kunjapur, Michael G. Napolitano, Eriona Hysolli, Karen Noguera, Evan M. Appleton, Max G. Schubert, Michaela A. Jones, Siddharth Iyer, Daniel J. Mandell, George M. Church
doi: https://doi.org/10.1101/2020.09.27.315804
Aditya M. Kunjapur
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
2Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, CLB 215, Newark, DE 19716, USA
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  • ORCID record for Aditya M. Kunjapur
  • For correspondence: kunjapur@udel.edu gchurch@genetics.med.harvard.edu
Michael G. Napolitano
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
3Ginkgo Bioworks, 27 Drydock Avenue, 8th Floor, Boston, MA 02210, USA
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Eriona Hysolli
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
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Karen Noguera
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
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Evan M. Appleton
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
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Max G. Schubert
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
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Michaela A. Jones
2Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, CLB 215, Newark, DE 19716, USA
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Siddharth Iyer
4Johns Hopkins University, 3101 Wyman Park Drive, Baltimore, MD 21218, USA
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Daniel J. Mandell
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
5GRO Biosciences, 700 Main Street North, Cambridge, MA 02139, USA
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George M. Church
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, MA 02115, USA
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  • For correspondence: kunjapur@udel.edu gchurch@genetics.med.harvard.edu
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Abstract

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications.

One Sentence Summary To ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

Competing Interest Statement

The authors declare the following competing financial interest(s): GMC has related financial interests in 64-x, EnEvolv, and GRO Biosciences. For a complete list of GMCs financial interests, please visit arep.med.harvard.edu/gmc/tech.html.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 28, 2020.
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Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells
Aditya M. Kunjapur, Michael G. Napolitano, Eriona Hysolli, Karen Noguera, Evan M. Appleton, Max G. Schubert, Michaela A. Jones, Siddharth Iyer, Daniel J. Mandell, George M. Church
bioRxiv 2020.09.27.315804; doi: https://doi.org/10.1101/2020.09.27.315804
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Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells
Aditya M. Kunjapur, Michael G. Napolitano, Eriona Hysolli, Karen Noguera, Evan M. Appleton, Max G. Schubert, Michaela A. Jones, Siddharth Iyer, Daniel J. Mandell, George M. Church
bioRxiv 2020.09.27.315804; doi: https://doi.org/10.1101/2020.09.27.315804

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