ERASynBIO:ECF Express - An Orthogonal, Organism-independent Expression Platform Based on Extractoplasmic Function (ECF) Sigma Factors

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The defining characteristics of synthetic biology are both the application of engineering principles to advance biological understanding and to develop new functions for useful purposes. One of the major challenges of synthetic biology is to modify or create new biological systems that are more efficient and reliable than natural organisms. This research project addresses one such challenge through the design and implementation of new regulatory modules that will control and optimize gene expression without wasting cellular resources. Significant broader impacts of this research are educating a next-generation workforce to support a growing bioeconomy, providing a new fundamental understanding of an important biological process, and generating new enabling tools for the wider synthetic biology community. This research takes advantage of a new process of using extracytoplasmic function sigma factors for synthetic biology applications. These extracytoplasmic function sigma factors (ECFs), when redesigned, can serve as robust gene regulatory switches capable of controlling diverse transcriptional activities. These factors have two conserved protein domains that recognize and bind to specific promoter sequences. They then recruit RNA-polymerase to initiate transcription from natural or synthetic promoter sites. The compositional simplicity of these factors are used for creating a series of synthetic sigma factor/promoter pairs tailored for specific regulatory needs. In this project, the investigators will create chimeric ECFs from natural ECFs to generate a large library of universal switches that are orthogonal in four model bacteria (E. coli, S. meliloti, B. subtilis and S. venezuelae). This will be accomplished by applying design strategies based on combinatorial synthesis, structure-guided mutational approaches and in vivo and in silico testing in complex genetic circuits. This results will provide new tools to enable biological design via synthetic biology. This project is the US collaborative component of a project funded through the ERASynBIO EU-US transnational funding mechanism.