Dual control of plasmid copy number in E. coli by combining temperature shifts and RNAI-based genetic circuits for tunable replication dynamics

Plasmid copy number (PCN) is a central determinant of recombinant gene expression levels and metabolic burden in bacterial cell factories. However, dynamic real-time control of PCN during microbial cultivation remains largely unexplored. In the ColE1/pUC19 replication system, PCN is governed by an antisense RNAI/RNAII circuit, but this native mechanism provides no external handle for process-level intervention. Here, we engineer and characterize a dual-layer control architecture that combines two orthogonal perturbations to direct plasmid replication in Escherichia coli populations i.e., temperature shifts, which modulate the secondary structure of RNAII and, consequently, its binding to RNAI and its inhibitory potency (ex vivo control), and arabinose-inducible overexpression of a supplementary RNAI cassette, integrated either on a secondary low-copy plasmid or directly on the high-copy target plasmid (in vivo control). Using a superfolder GFP validated fluorescent reporter and automated flow cytometry in continuous cultures, we track PCN dynamics at the population level in real time under chemostat conditions. Preliminary screening in microplates high-throughput cultivation device across three strains, two carbon sources, and three temperatures (30, 37, and 42C) reveals that the two control layers act synergistically. Neither temperature alone nor inducible RNAI alone achieves the dynamic range obtained by their combination. This synergistic effect is sustained during continuous cultivation at a fixed dilution rate of 0.1/h, with carbon source and temperature transitions producing predictable and reproducible shifts in population-level PCN. These results establish a scalable, dual-input framework for on-demand PCN tuning in continuous bioprocesses, with direct relevance to biopharmaceutical plasmid production and synthetic biology applications.