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. 2015 Jun 4;43(13):6511–6527. doi: 10.1093/nar/gkv584

Figure 2.

Figure 2.

Primer extension analysis of art200 in E. coli. (A) The DNA sequence of the 5' end of the art200 gene plus mutations introduced to deduce the −35/−10 promoter elements are shown. (B, C) Primer extension reactions performed to detect art200 from WT and the indicated mutant forms of tnpA-lacZ are shown. Total RNA was isolated from exponential phase cells grown in rich media (LB) and the primer (5'-end labeled with 32P) shown in Figure 1B was used to make cDNA. The cells contained IS200 on a multicopy plasmid. cDNAs were analyzed on a 10% denaturing polyacrylamide (sequencing) gel. The image in (B) shows a full gel; M = markers, U and C are RNA sequencing reactions. The image in (C) shows only the portion of a gel that includes the art200 primer extension products. For the latter, primer extension was multiplexed to include lpp as a loading control. The mutant PA-6 (bold letters) was used in subsequent experiments in this work to knock-down art200 levels in E. coli.