Fig. 5. Effects of G512 ori point mutations with and without DAS on plasmid copy number and on plasmid stability.

a. Effects on plasmid copy number. The plasmid copy number of pGFPuv containing point-mutations at the 512 position, both in the presence and absence of DAS, and of DAS_G628A ori was determined by miniprep extraction, restriction enzyme linearization and agarose gel electrophoresis as described in Methods. b. Quantitative measurement. Band intensities in the gel shown in section a were measured by densitometry using Image J software, with fixed area size for all the samples, and averaged. Error bars represent the standard deviation for three biological replicates. Statistically significant differences were confirmed by a 2-tailed, unpaired Student t test, with one asterisk indicative of p<0.05. c. Plasmid stability. The stability of the pGFPuv plasmids included in sections a and b was determined by serial passage in liquid culture in the absence of carbenicillin, and determining the % cells retaining carbenicillin resistance as described in Methods. Error bars represent the standard deviation of three biological replicates. The legend is shown on the upper right corner of the figure.