Fig. 1: Refactoring the pMB1 origin of replication.

A Schematic of the MB1 plasmid origin of replication (ORI) mechanism. The RNA primer is transcribed by RNA polymerase (RNAP) and, by default, folds into an RNA structure that is processed by RNase H to create a substrate for DNA polymerase I (Pol I) to initiate DNA replication. The antisense RNA is produced from an overlapping gene, which, if allowed to interact with the RNA primer, induces an alternative fold that does not initiate DNA replication. As the concentration of antisense RNA is proportional to the plasmid copy, this serves as a copy-control negative feedback loop. B Schematic of a refactored pMB1 ORI that separates the RNA primer and antisense RNA gene and introduces inactivating mutations in the P1 promoter sequence. C Shows sequences of mutations to inactivate the P1 promoter encoded inside the RNA primer. (Left graph) Whole-cell fluorescent characterization of the mutant promoters using a fluorescent reporter gene. (Right graph) Relative copy number of plasmids containing the original pMB1 ORI or refactored ORI with an RNA primer containing mutant P1 promoters. Copy number was characterized by encoding a constitutive RFP expression cassette onto the plasmid. Fluorescence characterization was performed (measured in units of fluorescence/optical density (OD) at 600 nm) in E. coli cells. Data show mean values ± SD and individual values of n = 4 biological replicates. Source data for this figure is available in the Source Data file.