Biochimie (2026). https://doi.org/10.1016/j.biochi.2026.08.008

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

Deschutter, M.S.; Beaumale, E.; Canaan, S.; Gago, G.; Santucci, P.

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.