Fig. 4. Effects of G180 ori point mutations (with and without DAS).

a. Effects on plasmid copy number. The copy number of pGFPuv plasmids containing G180 ori point mutations, both in the presence and absence of DAS, 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 replicates. Statistically significant differences were confirmed by a 2-tailed, unpaired Student t test, with one asterisk indicative of p<0.05 and two asterisks indicative of p<0.01. c. Plasmid stability of G180A-containing variants and d. Plasmid stability of G180C-containing variants. 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.