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Automated design of synthetic ribosome binding sites to control protein expression


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.

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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 ( 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.

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Authors and Affiliations



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.

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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).

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