Fig. 1: Design and characterization of the orthogonal transcription system.
a The binding of the orthogonal RNAP to its cognate promoters (with binding affinity KA) is a limiting step for protein synthesis in all organisms. The capping enzyme fused to RNAP ensures nuclear export and translation efficiency of the synthetic transcripts in mammalian cells. b The reporter gene expressions for the T7 promoter with RNAP-null, RNAP-only, RNAP-dead capping enzyme and RNAP-active capping enzyme. c Scheme of the quantitative measurement circuit composed of a monomeric RNA polymerase (RNAP) fused to a capping enzyme driven by the EF1α promoter and a set of T7 promoter variants in two separated plasmids. d Linearity between expression measured by flow cytometry and expression predicted by model in CHO cells transfected with 0.7 μg T7 RNAP plasmid. e Linearity between expression measured by flow cytometry and expression predicted by model in CHO cells transfected with 0.07 μg T7 RNAP plasmid. Data represent the mean ± SD (n = 3) f Comparison of expression from six different orthogonal promoters driven by cognate RNAPs and three widely-used mammalian promoters (CMV, EF1α and SV40) driven by the endogenous RNAP. Data represent the mean ± SD (n ≥ 6). g Characterization of a 6 × 6 orthogonality matrix of host-independent RNAPs and their cognate promoters. All data represent the mean ± SD (n ≥ 3). Source data are provided as a Source data file.
