Engineering E. coli strains using antibiotic-resistance-gene-free plasmids

We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning us-ing a dual auxotrophic-and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic-and essential-gene -based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a com-mon 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 long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad appli-cations while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.