Quantitation of CAT enzyme activity, CAT RNA level, and plasmid level in E. coli cells.

(A) Units of CAT activity detected in E. coli cell extract, containing different plasmid constructs, as indicated below the column graphs. No detectable CAT activity was found when E. coli 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 CAT 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 CAT 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. CAT 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 CAT WT construct is noted above each column. (B) Level of CAT RNAs estimated by RT-qPCR of the CAT RNA 3′-exon. This assay measures the sum of CAT pre-mRNA and CAT mRNA. (C) Molar amount of plasmid isolated from logarithmically growing E. coli 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 Figure 7, error bars denote standard deviations from three biological replicates.