Abstract
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.