An orthogonal, organism-independent expression platform based on extracytoplasmic function (ECF) sigma factors

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office

Abstract

Orthogonality is a key feature of classical engineering approaches but a major challenge to Synthetic Biology (SynBio) due to the high degree of context dependence and interconnectivity in biological systems. ECFexpress will develop a SynBio design framework based on Extracytoplasmic Function sigma factors (ECFs) to implement highly orthogonal regulatory switches and circuits. ECFs represent ideal building blocks for SynBio applications, because they are modular, inherently orthogonal, universal, and scalable. Initially, we will evaluate the organism-independent potential of ECFs by implementing orthogonal ECF-based regulation in four phylogenetically highly diverse bacteria, including two biotechnological workhorses. This will allow extracting and establishing universal design rules for choosing and implementing orthogonal ECF-based natural switches in any bacterium. Subsequently, we will design and engineer novel synthetic ECF switches with increased orthogonality. This goal will be achieved by applying design strategies based on combinatorial synthesis and structure-guided mutational approaches. Finally, we will use rational forward design to build and evaluate complex synthetic circuits based on ECF switches. This combined theoretical and experimental evaluation of novel ECF-based circuits will allow us to benchmark their orthogonality and to explore the ECF circuit design space. ECFexpress strictly adheres to engineering principles by (i) applying defined standards in assembly, measurements and data management, (ii) establishing orthogonal parts and switches for biological circuit design, and (iii) developing a bioinformatics platform for data management, mathematical modelling and forward design. Taken together, ECFexpress aims at a foundational advance by implementing true orthogonality to bacterial cells. It will be directly applicable to projects in basic research and in the knowledge-based bio-economy.

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