Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology

Abstract

AbstractEscherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, the EcN‐based toolbox was systematically expanded, enabling EcN as a powerful platform for more applications. First, two EcN cryptic plasmids and other compatible plasmids were genetically engineered to enrich the manipulable plasmid toolbox for multiple gene coexpression. Next, two EcN‐based technologies were developed, including the conjugation strategy for DNA transfer, and quantification of protein expression capability. Finally, the EcN‐based applications were further expanded by developing EcN native integrase‐mediated genetic engineering and establishing an in vitro cell‐free protein synthesis (CFPS) system. Overall, this study expanded the toolbox for manipulating and making full use of EcN as a commonly used probiotic chassis, providing several simplified, dependable, and predictable strategies for researchers working in synthetic biology fields.