Brief Communication | Published:

Predictable tuning of protein expression in bacteria

Nature Methods volume 13, pages 233236 (2016) | Download Citation

Abstract

We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expression level of any Escherichia coli gene by changing only a few bases. Measured protein levels for 91% of our designed sequences were within twofold of the desired target level.

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References

  1. 1.

    et al. Nat. Methods 10, 347–353 (2013).

  2. 2.

    et al. Nat. Methods 10, 354–360 (2013).

  3. 3.

    et al. Proc. Natl. Acad. Sci. USA 110, 14024–14029 (2013).

  4. 4.

    , & Science 342, 475–479 (2013).

  5. 5.

    et al. Nat. Chem. Biol. 8, 536–546 (2012).

  6. 6.

    , & Annu. Rev. Chem. Biomol. Eng. 4, 259–288 (2013).

  7. 7.

    et al. Nature 460, 894–898 (2009).

  8. 8.

    et al. ACS Synth. Biol. 4, 17–22 (2015).

  9. 9.

    , & Mol. Syst. Biol. 6, 360 (2010).

  10. 10.

    , & Cell 139, 1366–1375 (2009).

  11. 11.

    Annu. Rev. Biochem. 57, 199–233 (1988).

  12. 12.

    & Proc. Natl. Acad. Sci. USA 71, 1342–1346 (1974).

  13. 13.

    , & Nucleic Acids Res. 21, 4019–4023 (1993).

  14. 14.

    , , & J. Mol. Biol. 313, 215–228 (2001).

  15. 15.

    Methods Enzymol. 498, 19–42 (2011).

  16. 16.

    , , & Front. Bioeng. Biotechnol. 2, 1–6 (2014).

  17. 17.

    et al. Metab. Eng. 15, 67–74 (2013).

  18. 18.

    , & Nat. Biotechnol. 27, 946–950 (2009).

  19. 19.

    et al. Nucleic Acids Res. 42, W408–W415 (2014).

  20. 20.

    et al. Mol. Syst. Biol. 10, 731 (2014).

  21. 21.

    et al. Nat. Biotechnol. 22, 1567–1572 (2004).

  22. 22.

    , , & Nat. Biotechnol. 17, 691–695 (1999).

  23. 23.

    et al. Mol. Microbiol. 92, 1–9 (2014).

  24. 24.

    & Proc. Natl. Acad. Sci. U.S.A. 97, 6640–6645 (2000).

  25. 25.

    & Gene 158, 9–14 (1995).

  26. 26.

    , , , & Gene 111, 229–233 (1992).

  27. 27.

    et al. Nat. Biotechnol. 30, 521–530 (2012).

  28. 28.

    et al. Algorithms Mol. Biol. 6, 26 (2011).

  29. 29.

    & Biochem. Biophys. Res. Commun 290, 397–402 (2002).

  30. 30.

    , & Nat. Protoc. 2, 1896–1906 (2007).

  31. 31.

    , , , & Anal. Bioanal. Chem. 389, 1017–1031 (2007).

  32. 32.

    Nucleic Acids Res. 29, e45 (2001).

  33. 33.

    & J. Bacteriol. 186, 6714–6720 (2004).

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Acknowledgements

We thank H. Genee, A. Wallin, S. Cardinale and H. Wang for discussions and suggestions regarding this manuscript, and we thank A. Koza for assistance with DNA sequencing. The research leading to these results received funding from the Novo Nordisk Foundation through the Novo Nordisk Foundation Center for Biosustainability and the European Union Seventh Framework Programme (FP7-KBBE-2013-7-single-stage) under grant agreement 613745, Promys.

Author information

Author notes

    • Mads T Bonde
    • , Margit Pedersen
    •  & Michael S Klausen

    These authors contributed equally to this work.

Affiliations

  1. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.

    • Mads T Bonde
    • , Margit Pedersen
    • , Michael S Klausen
    • , Sheila I Jensen
    • , Tune Wulff
    • , Scott Harrison
    • , Alex T Nielsen
    • , Markus J Herrgård
    •  & Morten O A Sommer

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Contributions

M.T.B., M.P., M.S.K., S.I.J., T.W. and S.H. conducted the experiments. M.T.B., M.S.K. and M.J.H. conducted bioinformatics and data analysis. A.T.N. supervised the flow cytometry experiments. S.H. supervised the proteomics experiments. M.O.A.S., M.T.B., M.P. and M.S.K. designed the study. M.O.A.S. conceived and supervised the project. M.T.B., M.S.K. and M.O.A.S. wrote the manuscript, and all authors contributed to editing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Morten O A Sommer.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9

Excel files

  1. 1.

    Supplementary Table 1

    All 3,864 estimated GFP expression values.

  2. 2.

    Supplementary Table 2

    106 individually verified single colonies from the main library.

  3. 3.

    Supplementary Table 3

    EMOPEC designed oligos, ten expression levels for every annotated gene in E. coli K12 MG1655.

  4. 4.

    Supplementary Table 4

    Strains, plasmids and nucleotide oligos used in this study.

  5. 5.

    Supplementary Table 5

    Barcoded primers used for sequencing the flow-sorted bins.

  6. 6.

    Supplementary Table 6

    Sequence counts from the flow-sorted bins.

Zip files

  1. 1.

    Supplementary Software

    EMOPEC Python library. Source code for the EMOPEC algorithm as a Python library including the web server.

About this article

Publication history

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DOI

https://doi.org/10.1038/nmeth.3727

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