Construction of a <i>Mycobacterium smegmatis</i> Promoter Library for Therapeutic and Environmental Applications

<i>Mycobacterium smegmatis</i> 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 <i>M. smegmatis</i> 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 <i>M. smegmatis</i>, this study systematically identified and analyzed 18 <i>M. smegmatis</i> 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 <i>M. smegmatis</i> as an effective and field-deployable chassis.