Plasmids are a cornerstone of genetic engineering, but they are usually maintained in a cellular population by means of antibiotic selection pressure. Improper disposal of antibiotics and resistance genes from laboratories is an avenue for the spread of antibiotic resistance, and the use of antibiotic selection pressure is impossible in applications such as living diagnostics and therapeutics, environmental remediation, or large-scale bioproduction. Current efforts to mitigate the antibiotic dependence of genetic constructs require genomic integration and optimization or custom antibiotic-free plasmid maintenance systems that still rely on horizontally transferrable antibiotic resistance genes for initial plasmid construction. We addressed these shortcomings by creating a generalizable pipeline for antibiotic resistance gene-free plasmid (ARGFP) cloning and long-term maintenance, using a dual auxotrophic and essential gene-based selection strategy. We use auxotrophic selection to first construct plasmids in engineered E. coli DH10B cloning strains, and then both auxotrophic and essential gene-based selection to 1) select for recombinant strains and 2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory “wild-type” E. coli strain. We show that our approach has comparable efficiency to that of antibiotic resistance gene-based cloning. We also show that the double knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to plasmid maintenance for a month of repeated culturing as well as in the mouse gut, demonstrating the strong potential for broad applications in non-controlled environments without antibiotic resistance spread via horizontal gene transfer.

Gautam Dantas

Appearances