tRNA-Mediated Plasmid Stabilization for Antibiotic-Free Applications in <i>Escherichia coli</i>

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 <i>Escherichia coli</i> tryptophan tRNA (<i>trpT</i>) gene. We developed a workflow using a base strain with a <i>trpT</i> deletion and a temperature-sensitive <i>trpT</i>-expressing plasmid to circumvent the need for remaking chromosomal <i>trpT</i> deletions for every transformation. We evaluated the stability of a range of antibiotic gene-free <i>trpT</i> 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 <i>trpT</i>, 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.