Investigations of convergent and divergent synthesis of Dome11 dsRNA in vivo within E. coli. Plasmids D11_C, D11_D, D11_D_1kb, and D11_D_2kb were investigated. (A) Growth curve comparison of E. coli HT115 cells transformed with each of the plasmids. An outgrowth was performed and allowed to grow to an OD600 of 0.4–0.6. Samples were then induced for 4 h, with OD measurements recorded at hour time points. Data shown as mean ± SD and are representative of three biological replicates. (B) Agarose gel electrophoresis analysis of RNA extractions in the absence and presence of RNase T1. The corresponding rRNA and Dome11 dsRNA (400 bp) are highlighted. (C) IP-RP HPLC chromatography analysis in the absence and presence of RNase T1. (D) Quantification of dsRNA yield. RNA extractions were performed in the presence of RNase T1. RNA samples were analyzed using UV spectrophotometry to determine RNA concentration using a mass concentration/A260 unit (46.52 μg/mL). Significance was calculated using a one-way ANOVA test with multiple comparisons. Convergent-produced Dome11 (pD11_C) demonstrates the highest absolute yield of 36.95 µg. Data are shown as box plots of triplicate technical replicates and are representative of three biological replicates; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.
