Antibiotic-Free Plasmid Selection and Maintenance in Bacteria

Abstract

The use of antibiotics and antibiotic resistance genes is rapidly becoming unacceptable in many areas of biotechnology, such as recombinant protein production, engineering of transgenic organisms, DNA vaccine and gene therapy applications. Plasmid-borne antibiotic resistant genes cause a considerable metabolic burden to the host bacterial cell. The resistance gene product, or even residual antibiotic contamination, can induce an immune response or cytotoxicity in patients during therapeutic applications. The risk of antibiotic resistant genes spreading from genetically-modified organisms to environmental pathogens is also of concern. Here we discuss the alternative methods of maintaining recombinant plasmids in bacteria. These include the complementation of a host auxotrophy, post-segregational killing mechanisms, the generation of minicircles by recombination, and the technique that we have developed involving the use of repressor titration. Some of these systems use antibiotics for selection of transformants but have alternative mechanisms of plasmid maintenance, whilst others are completely independent of antibiotics and their resistance genes.