tRNA-Mediated Plasmid Stabilization for Antibiotic-Free Applications in Escherichia coli

Plasmids are essential tools in molecular biology and biotechnology. In research laboratories, it is common to use antibiotic selection markers to ensure that plasmids are stably maintained in a cellular population. However, the use of antibiotics poses a significant challenge in the industrial scale-up process due to the high cost and the risk of spreading resistance. Therefore, methods for antibiotic-free plasmid maintenance are in high demand. Here, we present an essential gene-based plasmid selection strategy utilizing the Escherichia coli tryptophan tRNA (trpT) gene. We developed a workflow using a base strain with a trpT deletion and a temperature-sensitive trpT-expressing plasmid to circumvent the need for remaking chromosomal trpT deletions for every transformation. We evaluated the stability of a range of antibiotic gene-free trpT plasmids with different copy numbers and determined that the system is as efficient as, or better than, systems using antibiotics. Furthermore, the system is stable when producing a biochemical at industrially relevant fermentation conditions, and due to the small size of trpT, it allows for plasmid minimization. The approach constitutes a significant contribution toward developing simpler and more effective antibiotic-free bioprocesses and combating the spread of multiresistant infections.