A simple multi-cloning strategy for gene expression and subcellular localization in mycobacteria

Genetic manipulation of mycobacteria remains critical in functional genomics and modern microbiology. However, this process is often laborious due to limited cloning flexibility and low throughput. Here, we describe a streamlined cloning platform for expressing a gene of interest either alone or fused to a monomeric fluorescent protein under the control of two promoters of choice. This platform combines three simple PCR amplification reactions and four simultaneous ligations. Accordingly, this four-step procedure provides a fast and flexible cloning strategy for protein expression and subcellular localization analyses in mycobacteria. This cost-effective approach is compatible with some commonly used mycobacterial vectors and host strains. Using M. tuberculosis triacylglycerol synthase 1 gene (tgs1) as a proof of concept, we demonstrate the efficient cloning, robust expression, and reliable localization of the resulting fusion protein. This platform significantly reduces hands-on time and experimental complexity, providing a practical tool for functional and microbiological studies in mycobacteria.