A Plasmid System with Tunable Copy Number

Plasmids are one of the most commonly used and time-tested molecular biology platforms for synthetic biology and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we demonstrate two methods we've developed that allow for finely-tuned control of ColE1 plasmid copy number: a plasmid with an anhydrotetracycline-inducible copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. These systems are used to probe the effects of plasmid and gene copy number on cellular growth rates, gene expression, biosynthetic pathway production, and the behavior of a simple genetic circuit. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on variability in gene expression. Using our massively parallel assay, we find that plasmids impose a linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as a tool for the optimization of synthetic biological systems.