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Automated design of synthetic ribosome binding sites to control protein expression

Nature Biotechnology volume 27, pages 946–950 (2009)Cite this article

Abstract

Microbial engineering often requires fine control over protein expression—for example, to connect genetic circuits1,2,3,4,5,6,7 or control flux through a metabolic pathway8,9,10,11,12,13. To circumvent the need for trial and error optimization, we developed a predictive method for designing synthetic ribosome binding sites, enabling a rational control over the protein expression level. Experimental validation of >100 predictions in Escherichia coli showed that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing protein expression to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach should accelerate the construction and systematic optimization of large genetic systems.

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Figure 1: A thermodynamic model of bacterial translation initiation.
Figure 2: A ribosome binding site design method.
Figure 3: The RBS design method can control the expression level of different proteins by accounting for the influence of the protein coding sequence.
Figure 4: Optimal connection of a sensor input to an AND gate genetic circuit.

References

  1. Basu, S., Gerchman, Y., Collins, C.H., Arnold, F.H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature 434, 1130–1134 (2005).

    Article  CAS  Google Scholar 

  2. Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008).

    Article  CAS  Google Scholar 

  3. Friedland, A.E. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).

    Article  CAS  Google Scholar 

  4. Ellis, T., Wang, X. & Collins, J.J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat. Biotechnol. 27, 465–471 (2009).

    Article  CAS  Google Scholar 

  5. Yokobayashi, Y., Weiss, R. & Arnold, F.H. Directed evolution of a genetic circuit. Proc. Natl. Acad. Sci. USA 99, 16587–16591 (2002).

    Article  CAS  Google Scholar 

  6. Tabor, J.J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).

    Article  Google Scholar 

  7. Anderson, J.C., Voigt, C.A. & Arkin, A.P. Environmental signal integration by a modular AND gate. Mol. Syst. Biol. 3, 133 (2007).

    Article  Google Scholar 

  8. Dueber, J.E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).

    Article  CAS  Google Scholar 

  9. Anthony, J.R. et al. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metab. Eng. 11, 13–19 (2008).

    Article  Google Scholar 

  10. Atsumi, S., Hanai, T. & Liao, J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008).

    Article  CAS  Google Scholar 

  11. Hawkins, K.M. & Smolke, C.D. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 4, 564–573 (2008).

    Article  CAS  Google Scholar 

  12. Lee, K.H., Park, J.H., Kim, T.Y., Kim, H.U. & Lee, S.Y. Systems metabolic engineering of Escherichia coli for L-threonine production. Mol. Syst. Biol. 3, 149 (2007).

    Article  CAS  Google Scholar 

  13. Lutke-Eversloh, T. & Stephanopoulos, G. Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression. Metab. Eng. 10, 69–77 (2008).

    Article  Google Scholar 

  14. Czar, M.J., Anderson, J.C., Bader, J.S. & Peccoud, J. Gene synthesis demystified. Trends Biotechnol. 27, 63–72 (2009).

    Article  CAS  Google Scholar 

  15. Gibson, D.G. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 (2008).

    Article  CAS  Google Scholar 

  16. Isaacs, F.J. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat. Biotechnol. 22, 841–847 (2004).

    Article  CAS  Google Scholar 

  17. Carrier, T.A. & Keasling, J.D. Library of synthetic 5′ secondary structures to manipulate mRNA stability in Escherichia coli. Biotechnol. Prog. 15, 58–64 (1999).

    Article  CAS  Google Scholar 

  18. Pfleger, B.F., Pitera, D.J., Smolke, C.D. & Keasling, J.D. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027–1032 (2006).

    Article  CAS  Google Scholar 

  19. Chubiz, L.M. & Rao, C.V. Computational design of orthogonal ribosomes. Nucleic Acids Res. 36, 4038–4046 (2008).

    Article  CAS  Google Scholar 

  20. de Smit, M.H. & van Duin, J. Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc. Natl. Acad. Sci. USA 87, 7668–7672 (1990).

    Article  CAS  Google Scholar 

  21. Vellanoweth, R.L. & Rabinowitz, J.C. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol. Microbiol. 6, 1105–1114 (1992).

    Article  CAS  Google Scholar 

  22. Xia, T. et al. Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 37, 14719–14735 (1998).

    Article  CAS  Google Scholar 

  23. Mathews, D.H., Sabina, J., Zuker, M. & Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    Article  CAS  Google Scholar 

  24. Kierzek, R., Burkard, M.E. & Turner, D.H. Thermodynamics of single mismatches in RNA duplexes. Biochemistry 38, 14214–14223 (1999).

    Article  CAS  Google Scholar 

  25. Miller, S., Jones, L.E., Giovannitti, K., Piper, D. & Serra, M.J. Thermodynamic analysis of 5′ and 3′ single- and 3′ double-nucleotide overhangs neighboring wobble terminal base pairs. Nucleic Acids Res. 36, 5652–5659 (2008).

    Article  CAS  Google Scholar 

  26. Christiansen, M.E. & Znosko, B.M. Thermodynamic characterization of the complete set of sequence symmetric tandem mismatches in RNA and an improved model for predicting the free energy contribution of sequence asymmetric tandem mismatches. Biochemistry 47, 4329–4336 (2008).

    Article  CAS  Google Scholar 

  27. Laursen, B.S., Sorensen, H.P., Mortensen, K.K. & Sperling-Petersen, H.U. Initiation of protein synthesis in bacteria. Microbiol. Mol. Biol. Rev. 69, 101–123 (2005).

    Article  CAS  Google Scholar 

  28. Studer, S.M. & Joseph, S. Unfolding of mRNA secondary structure by the bacterial translation initiation complex. Mol. Cell 22, 105–115 (2006).

    Article  CAS  Google Scholar 

  29. Chen, H., Bjerknes, M., Kumar, R. & Jay, E. Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res. 22, 4953–4957 (1994).

    Article  CAS  Google Scholar 

  30. Kudla, G., Murray, A.W., Tollervey, D. & Plotkin, J.B. Coding-sequence determinants of gene expression in Escherichia coli. Science 324, 255–258 (2009).

    Article  CAS  Google Scholar 

  31. de Smit, M.H. & van Duin, J. Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA. J. Mol. Biol. 331, 737–743 (2003).

    Article  CAS  Google Scholar 

  32. Dirks, R.M., Bois, J.S., Schaeffer, J.M., Winfree, E. & Pierce, N.A. Thermodynamic Analysis of Interacting Nucleic Acid Strands. SIAM Rev. 49, 65–88 (2007).

    Article  Google Scholar 

  33. Sengupta, J., Agrawal, R.K. & Frank, J. Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA. Proc. Natl. Acad. Sci. USA 98, 11991–11996 (2001).

    Article  CAS  Google Scholar 

  34. David, F., Hagendorf, C. & Wiese, K.J. A growth model for RNA secondary structures. J. Stat. Mech. Theor. Exp. P04008 (2008).

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Acknowledgements

We are grateful to all members of the Voigt lab for technical advice and continued support. This work is supported by the Pew and Packard Foundations, Office of Naval Research, National Institutes of Health (NIH) EY016546, NIH AI067699, NSF BES-0547637, National Science Foundation (NSF) TeraGrid TG-MCB080126T and a Sandler Family Opportunity Award. C.A.V., H.M.S., and E.A.M. are part of the NSF SynBERC Engineering Research Center (http://www.synberc.org/). E.A.M. is supported by an NSF Graduate Research Fellowship and an American Society for Engineering Education National Defense Science and Engineering Graduate Fellowship.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA

    Howard M Salis & Christopher A Voigt

  2. Graduate Group in Biophysics, University of California San Francisco, San Francisco, California, USA

    Ethan A Mirsky

Authors
  1. Howard M Salis
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  2. Ethan A Mirsky
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Contributions

H.M.S and C.A.V designed the study and wrote the manuscript. H.M.S. developed the method. H.M.S. and E.A.M. performed the experiments.

Corresponding author

Correspondence to Christopher A Voigt.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–11, Supplementary Discussion and Supplementary Methods (PDF 2317 kb)

Supplementary Table I

A table of all ribosome binding site sequences created in this study, their predicted Gtot, their measured protein expression levels, and doubling times. (XLS 89 kb)

Supplementary Data (TXT 7 kb)

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Salis, H., Mirsky, E. & Voigt, C. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27, 946–950 (2009). https://doi.org/10.1038/nbt.1568

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