Quantitation of CAT enzyme activity, <i>CAT</i> RNA level, and plasmid level in <i>E. coli</i> cells.

<p>(<b>A</b>) Units of CAT activity detected in <i>E. coli</i> cell extract, containing different plasmid constructs, as indicated below the column graphs. No detectable CAT activity was found when <i>E. coli</i> cells contained the plasmid pUC19 (left black column; -0.00045±0.00053 units per OD₆₀₀). Grey columns show the CAT activity of constructs expressing a spliceozyme and a <i>CAT</i> pre-mRNA containing an intron that mediated efficient bacterial resistance to chloramphenicol. White columns show the CAT activity mediated by introns that led to poor or no bacterial resistance to chloramphenicol. Construct names starting with 64 or 100 label the length of the intron in the <i>CAT</i> pre-mRNA gene, ‘S’ denotes that the clone was selected for activity on medium with chloramphenicol, ‘L’ denotes that the clone was chosen from an un-selected library, ‘I’ denotes that the library for this selection was randomized in the internal region of the intron, and the number after C is the clone number. <i>CAT</i> WT denotes a construct without spliceozyme, and in which the CAT gene did not contain an intron, resulting in 0.13±0.01 units of CAT activity per OD₆₀₀. The percent of CAT activity relative to the <i>CAT</i> WT construct is noted above each column. (<b>B</b>) Level of <i>CAT</i> RNAs estimated by RT-qPCR of the <i>CAT</i> RNA 3′-exon. This assay measures the sum of <i>CAT</i> pre-mRNA and <i>CAT</i> mRNA. (<b>C</b>) Molar amount of plasmid isolated from logarithmically growing <i>E. coli</i> cells. Note that the unit OD₆₀₀ is used as an absolute value, corresponding to the cells in a volume of 1 mL cell suspension with OD₆₀₀ = 1. For all graphs shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101932#pone-0101932-g007" target="_blank">Figure 7</a>, error bars denote standard deviations from three biological replicates.