A robust Escherichia coli cell-free expression toolbox driven by sigma factors

Abstract

The development of various expression systems leads to wide and exciting applications of cell-free protein synthesis (CFPS) in synthetic biology. However, the limited regulatory transcription elements still restrict the applications. This work presents a set of orthogonal expression components with various expression features in the Escherichia coli-based cell-free systems. These expression systems were based on the endogenous sigma factors from E. coli and heterologous sigma factors from Bacillus subtilis that naturally recognize specific promoter sequences with different strength. Sigma factors and their specific promoters were combined and screened to function orthogonally between each other in E. coli-based cell-free systems. Additionally, several promoters were confirmed constitutive expression in cell-free systems, which can be used as the primary transcription in the regulatory cascades for gene circuits, getting rid of the bacteriophage expression system. Promoters were performed and orthogonally regulated by sigma factors in varying expression strength, which enlarges the expression elements for cell-free systems. The sigma factor toolbox contributes to the assembly of more complex synthetic genetic circuits in cell-free systems and could constitute an exciting expansion in synthetic biology.