Figure 1. See also Table 2. Cas3 promotes ColE1 plasmid copy number. (A) Yields of the ColE1-based plasmid pUC19 were measured after extraction from E. coli MG1655 cells. Cells contained either pUC19 as empty plasmid vector, or pUC19 expressing Cas3, Cas3^K320LL (helicase defective), Cas3^K78L (nuclease defective), Cascade or Cas7 as indicated. Results are means of three independent tests with standard deviation error bars. The panel shows a typical outcome of gel electrophoresis loading 4 μl of uncut plasmid extracts (pUC, pCas3 or pCas3^K320LL) in an ethidium bromide-stained agarose gel (0.8%). Arrows within the pCas3 lane point to slow-migrating plasmid multimers (see later Results section). Supercoiled plasmid is arrowed (SC); note that cas3 adds 2.9 kb to the size of pUC19, accounting for slower migration of pCas3 SC species compared with SC pUC19. No DNA size ladder is present because plasmids are uncut. (B) Yields of plasmids based on pRSF-1b or pACYC-Duet were measured after extraction from, respectively, BL21 AI or strain IIB967. pRSF-1b or PACYC-Duet were either empty plasmid, or expressed Cas3, as indicated. Arabinose (+ ara) was used to induce T7 polymerase that is needed to transcribe cas3 in each plasmid. Glucose (+ glu) gives tight repression of T7 polymerase. Results are means of two independent tests with error bars for standard deviation from the mean.