Investigations of convergent and divergent synthesis of GFP, Sec23A, and 120 bp dsRNA in vivo within E. coli. Plasmids GFP_C, GFP_D, Sec23A_C, Sec23A_D, 120_C, and 120_D 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 GFP (263 bp), Sec23A (1504 bp), and 120 bp dsRNA are highlighted. (C) IP-RP HPLC chromatography analysis in the absence and presence of RNase T1. Insert is shown for Sec23A dsRNA in the absence 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 unpaired t-tests, against each gene sequence pairing. No significant difference was noted between GFP constructs. Convergent-produced Sec23A (pSec23A_C) demonstrated the highest absolute yield (17.53 µg) between Sec23A constructs. Divergent-produced 120 bp (p120_D) demonstrated the highest absolute yield (9.29 µg) between 120 bp constructs. 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.
