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Metagenomic mining of regulatory elements enables programmable species-selective gene expression

  • Nature Methods volume 15, pages 323329 (2018)
  • doi:10.1038/nmeth.4633
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Abstract

Robust and predictably performing synthetic circuits rely on the use of well-characterized regulatory parts across different genetic backgrounds and environmental contexts. Here we report the large-scale metagenomic mining of thousands of natural 5′ regulatory sequences from diverse bacteria, and their multiplexed gene expression characterization in industrially relevant microbes. We identified sequences with broad and host-specific expression properties that are robust in various growth conditions. We also observed substantial differences between species in terms of their capacity to utilize exogenous regulatory sequences. Finally, we demonstrate programmable species-selective gene expression that produces distinct and diverse output patterns in different microbes. Together, these findings provide a rich resource of characterized natural regulatory sequences and a framework that can be used to engineer synthetic gene circuits with unique and tunable cross-species functionality and properties, and also suggest the prospect of ultimately engineering complex behaviors at the community level.

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Acknowledgements

We thank members of the Wang lab for helpful discussions and feedback. H.H.W. acknowledges funding support from the NIH (1DP5OD009172-02, 1U01GM110714-01A1), NSF (MCB-1453219), Sloan Foundation (FR-2015-65795), DARPA (W911NF-15-2-0065), and ONR (N00014-15-1-2704). N.I.J. is supported by an NSF Graduate Research Fellowship (DGE-16-44869). S.S.Y. is supported by the National Research Foundation of Korea (NRF-2017R1A6A3A03003401). We also thank T. Seto for help with plasmid construction; A. Figueroa for assistance with cell sorting; H. Salis for helpful discussions regarding the RBS calculator; D.B. Goodman for discussions regarding FACS-seq; G.M. Church (Harvard Medical School, Boston, Massachusetts, USA) for access to OLS libraries; and D. Dubnau (Rutgers New Jersey Medical School, Newark, New Jersey, USA), S. Lory, and A. Rasouly (both at Harvard Medical School, Boston, Massachusetts, USA) for providing the BD3182 and PAO1 Δpsy2 strains.

Author information

Author notes

    • Antonio L C Gomes

    Present address: Department of Immunology, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Nathan I Johns
    •  & Antonio L C Gomes

    These authors contributed equally to this work.

Affiliations

  1. Department of Systems Biology, Columbia University Medical Center, New York, New York, USA.

    • Nathan I Johns
    • , Antonio L C Gomes
    • , Sung Sun Yim
    • , Tomasz Blazejewski
    •  & Harris H Wang
  2. Integrated Program in Cellular, Molecular and Biomedical Studies, Columbia University Medical Center, New York, New York, USA.

    • Nathan I Johns
    •  & Tomasz Blazejewski
  3. School of Engineering and Applied Sciences, Columbia University, New York, New York, USA.

    • Anthony Yang
  4. Broad Institute, Cambridge, Massachusetts, USA.

    • Christopher S Smillie
    •  & Eric J Alm
  5. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Mark B Smith
    •  & Eric J Alm
  6. Computational and Systems Biology Initiative, MIT, Cambridge, Massachusetts, USA.

    • Eric J Alm
  7. The Center for Microbiome Informatics and Therapeutics, MIT, Cambridge, Massachusetts, USA.

    • Eric J Alm
  8. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, USA.

    • Sriram Kosuri
  9. UCLA–DOE Institute for Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California, USA.

    • Sriram Kosuri
  10. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA.

    • Sriram Kosuri
  11. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, USA.

    • Harris H Wang

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Contributions

N.I.J., A.L.C.G., C.S.S., M.B.S., E.J.A., S.K., and H.H.W. designed the study. N.I.J., S.S.Y., and H.H.W. performed the experiments. N.I.J., A.L.C.G., A.Y., T.B., and H.H.W. analyzed the data. N.I.J., A.L.C.G., and H.H.W. wrote the manuscript, with input from all other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Harris H Wang.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Regulatory sequence library metadata

  2. 2.

    Supplementary Table 2

    Library expression data for B. subtilis, E. coli, and P. aeruginosa

  3. 3.

    Supplementary Table 3

    Library expression data for E. coli in five growth conditions

  4. 4.

    Supplementary Table 4

    RS241 library expression data in six species

  5. 5.

    Supplementary Data Set 1

    Strains and materials used in this study