Figure 3. Gene silencing in E. coli to obtain a titration of declining growth rates and transcript levels by antisense PNA and expressed antisense.

E. coli AS19 was treated with PNA doses, while TOP10 clones containing antisense-expressing plasmids were induced with various IPTG concentrations as shown in Figure 2, so as to obtain growth inhibition in a dose-dependent manner. Total RNA from E. coli cultures was harvested at the time when the control culture increased in OD₅₅₀ by 0.1. RNA was used for cDNA synthesis and quantitative real-time PCR to determine mRNA levels by normalization to a reference gene rpoA, and then calculated relative to the unsilenced (untreated) control. Growth rates were determined by OD₅₅₀ readings over time. The relationship between mRNA and growth rate decline indicated growth requirement stringency of each gene.