Plasmid copy number control offers a versatile tool in synthetic biology applications.

Plasmids are typically regarded as static delivery vehicles for foreign DNA. Here, we expose the pivotal role that plasmid copy number (PCN) control can play in synthetic biology, not only in Escherichia coli but also in the next-generation bacterial workhorse Vibrio natriegens. We show that the antibiotic selection marker can impact PCN, thus affecting growth and protein production, and that cells can be cotransformed with multiple variants of the same plasmid, with their PCN controlled simultaneously and in unison. We reveal that plasmid loss can be mitigated via the integration of an additional origin of replication (ori) and that PCN control can be leveraged to modulate horizontal gene transfer, which we illustrate within the context of conjugation-based intercellular communication. Finally, we expand the MoClo modular cloning framework with inducible PCN control for rapid prototyping and to enhance the performance of complex biocircuits.