Construction and Characterization of a Tunable Plasmid Copy Number System

Plasmids have evolved elegant mechanisms to ensure stable replication which balance the probability of plasmid loss with their metabolic burden on host cells. The pUC19 plasmid controls its copy number through a replication priming RNA (RNAII) and an inhibitory antisense RNA (RNAI). Through randomized mutagenesis of the promoters controlling these RNAs, we have created a library of over 1000 plasmids with copy numbers spanning a range greater than 2 orders of magnitude, allowing plasmid copy number to be used as a tunable parameter in several systems. We demonstrate the broad applicability of this system by using it to tune the behavior of simple genetic circuit elements and determine the fitness cost of green fluorescent protein and several CRISPR proteins. We additionally infer transcription rates for over 1000 different promoters of both RNAI and RNAII by combining our copy number measurements with the results of stochastic molecular simulations.