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.质粒（Plasmid）在分子生物学与生物技术领域应用广泛，但宿主细胞内的稳定性缺陷会制约其稳健复制。本研究系统表征了大肠杆菌（Escherichia coli）在每细胞3~586个质粒拷贝数、多种质粒尺寸、不同筛选标记，以及葡萄糖、甘油两种培养基条件下，针对质粒负荷的转录与生理响应。我们利用机器学习算法从转录组学数据中提取共调控基因模块，鉴定出SOS应激反应（SOS stress response）为质粒负荷的特征性响应。SOS反应的激活程度与质粒拷贝数显著相关，而非额外DNA总含量，且会通过上调分裂抑制剂sulA引发细胞丝状化。本研究借助流式细胞术（flow cytometry）检测细胞尺寸与细胞复杂度，证实细胞丝状化与细胞大小异质性与SOS反应的激活密切相关。研究结果表明，质粒不稳定性源于复制频率驱动的细胞应激，而非代谢负荷，并指出SOS反应是提升质粒稳定性的关键干预靶点。