Lessons from enzymology: How do ribosome binding sites affect affinity and basal expression in inducible genetic devices?

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Max Carbonell-Ballestero, Salvador Duran-Nebreda, Javier Macia and Carlos Rodriguez-Caso

ICREA-Complex Systems Lab, Spain

It is widely assumed that the strength of ribosome binding sites (RBSs) is one of the most relevant issues in the experimental implementation of synthetic devices, often involving a trade-off between basal signal due to leakiness and output range amplitude. Although models of regulable promoters exist, there is still a lack of knowledge on how should systems be engineered in order to display given, predefined characteristics. In this work we present a simple mathematical approach that allows us to understand the design principles of an inducible promoter. As a proof of concept, our theoretical results are contrasted with empirical data by constructing a library of genetic devices, expressing a Red Fluorescence Protein (RFP) gene reporter by the control of a Lux promoter of Vibrio fischeri in E. coli. In this device, the expression of the homoserine-lactone receptor is constitutively produced but modulated by using different RBSs differing in their strength. Using Biobrick cloning assembly and the part registry repository, we characterize the effects of changing RBSs in both gene reporter and homoserine-lactone receptor. The analysis of transfer functions of the library shows that the use of a higher RBS strength at the gene codifying the Lux receptor produced an increase of the input affinity without a marked enhance of the basal output. However, the increase of the RBS at the reporter gene contributed to enhance the output but, with a penalty on increasing the basal level. Using our case study as a reference, we propose a set of guidelines to better design synthetic circuits and achieve more desirable system’s behaviour.