Preventing T7 RNA Polymerase Read-through Transcription—A Synthetic Termination Signal Capable of Improving Bioprocess Stability

Abstract

The phage-derived T7 RNA polymerase is the most prominent orthogonal transcriptions system used in the field of synthetic biology. However, gene expression driven by T7 RNA polymerase is prone to read-through transcription due to contextuality of the T7 terminator. The native T7 terminator has a termination efficiency of approximately 80% and therefore provides insufficient insulation of the expression unit. By using a combination of a synthetic T7 termination signal with two well-known transcriptional terminators (rrnBT1 and T7), we have been able to increase the termination efficiency to 99%. To characterize putative effects of an enhanced termination signal on product yield and process stability, industrial-relevant fed batch cultivations have been performed. Fermentation of a E. coli HMS174(DE3) strain carrying a pET30a derivative containing the improved termination signal showed a significant decrease of plasmid copy number (PCN) and an increase in total protein yield under standard conditions.

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

“…The bicistronic expression cassettes were structured identically (Figure ). They were composed of the untranslated region (UTR) 5′‐UTR with the T7 promoter/lac operator element (T7 p/o) and a ribosome‐binding site (RBS) for the LC, the LC with signal sequence (LC SS ), the UTR linker with a spacer and a RBS for the HC, the HC with signal sequence (HC SS ), and the transcription terminator tZenit . The resulting eight combinations were first cloned into the pET30a vector between the Nde I and the Eco RI restriction sites; then, the vectors were transformed into E. coli host strains, BL21(DE3) and HMS174(DE3).…”

Codon usage influences fitness through RNA toxicity

“…To remove possible out-of-frame translation, we inserted stop codons into GFP_170 in all three frames, before and after the toxic region, and toxicity remained the same in all cases (Supp Figure 4C). Third, we introduced an efficient synthetic T7 transcription terminator 20 upstream of the toxic region in GFP_170 and in the corresponding location in GFP_012. Notably, we found that both variants with internal transcription terminators became nontoxic, and GFP_170_TT grew slightly faster than GFP_012_TT (Figure 2D).…”

Synthetic Biology: Computational Modeling Bridging the Gap between In Vitro and In Vivo Reactions

“…In this sense, a library containing more than 500 terminators was characterized in E. coli, and the strengths of the terminators were in line with predicted strengths based on a simple thermodynamic model [105]. In addition, T7-RNAP terminators with almost the maximum efficiency have been developed [106]. These models were tested in vivo.…”