Construction of a Mycobacterium smegmatis Promoter Library for Therapeutic and Environmental Applications

Mycobacterium smegmatis is a nonpathogenic species of soil-dwelling mycobacteria that shows promise as a synthetic biology chassis with both clinical and environmental applications. The development of a nonmodel chassis requires a library of regulatory genetic elements that cover a range of expression levels. Currently, most studied M. smegmatis promoters are characterized using single-channel reporter cassettes, which are vulnerable to extrinsic noise introduced by different culturing conditions, initial cell metabolic states, and reporter genes of choice. For constructing predictable and reliable circuits in M. smegmatis, this study systematically identified and analyzed 18 M. smegmatis promoters by using a dual-channel reporter system across different environments. Here, we show a well-characterized promoter library and a standardizable reporter plasmid construct that will allow future investigators to easily assess additional promoter elements, promoting future use of M. smegmatis as an effective and field-deployable chassis.