Automated design of synthetic ribosome binding sites to control protein expression

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Abstract
Salis et al. design precisely tuned ribosome binding sites that allow rational control over the rate of protein translation. This technology should facilitate the design of synthetic genetic circuits and metabolic pathways. 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.