Exploring the Performance Space of a Genetic Toggle Switch across Host and RBS context

Abstract

Synthetic biology applies engineering principles onto biological systems to rationally create novel devices with desired properties. The behavior of an engineered genetic circuit is influenced by a complex multitude of factors, including regulatory elements and the cellular environment it operates within. This thesis delves into the impact of ribosome binding site (RBS) configurations and host context on shaping the performance space of a synthetic genetic toggle switch. A total of nine toggle switches were assembled using the BASIC DNA assembly platform, incorporating different combinations of three RBS strengths regulating the translation efficiency of the transcriptional repressors. Analysis of quantifiable performance parameters revealed differing performance profiles across toggle switches with varying RBS combinations. Moreover, this work investigated the impact of host-context on toggle switch performance (i.e. the chassis-effect) by transforming the genetic circuits into Escherichia coli DH5α and Pseudomonas putida KT2440. Strong clustering of performance profiles was observed along host organisms, suggesting a clear chassis-effect dominating over RBS context in driving toggle switch performance. This work unveils the opportunity of the chassis-effect to expand the design space of a genetic circuit, underlining the pivotal role of selecting an appropriate chassis as a crucial variable in synthetic biodesign. Furthermore, harnessing the fine-tuning potential of RBS can serve as an efficient strategy to regulate and optimize gene expression levels. Considering a multitude of design parameters that allow for strategical tuning will pave the way for precise control over gene expression, enhancing the functionality of genetic circuits, and expanding the repertoire of tools available for synthetic biology endeavors.