Raw flow cytometry dataset on E. coli plasmid hosts.

Plasmids are widely used across molecular biology and biotechnology, but robust replication is limited by stability issues in host cells. Here we systematically characterized transcriptional and physiological responses to plasmid burden in Escherichia coli across 3–586 copies per cell, multiple plasmid sizes, selection markers, in both glucose and glycerol. Using a machine-learning algorithm to extract co-regulated gene modules from transcriptomics data, we identified the SOS stress response as a signature of plasmid burden. The activation of the SOS response correlated strongly with the number of plasmid copies rather than total additional content of DNA, and resulted in cell filamentation through upregulation of the division inhibitor sulA. Using flow cytometry to measure cell size and complexity, we show that cell filamentation and cell size heterogeneity correlates with activation of the SOS response. These results demonstrate that plasmid instability arises from cellular stress driven by replication frequency rather than metabolic burden, and point to the SOS response as a key target for improving plasmid stability.