Figure 2: The TRAP-tbs configuration confers a translational block resulting in potent repression of GFP expression in HEK293T cells.
(a) The GFP reporter plasmid containing the tbs within the 5′UTR of the transcription unit and the TRAP-expression plasmid used throughout the study. B. subtilis TRAP was codon-optimised for human cell expression and C-terminally Hisx6 tagged. (b) 2-D plots (FSC v FL1/GFP) of HEK293T cells co-transfected with GFP reporter plasmids and TRAP or control plasmids, analysed by flow cytometry (MFI, median fluoresence intensity [arbitrary units]). (c) GFP Expression scores for the co-transfections were generated (percent GFP × MFI; plotted on left y-axis) and qRT-PCR data of cytoplasmic GFP RNA copies detected plotted on the right y-axis; grey bars—RT-positive, white bars—no RT (residual pDNA control). All data are mean average values±s.d. [log10-transformed data] (n=8); *P<1.7 × 10−17 [Welch’s t-test]. (d) Fold repression of GFP Expression Scores (difference in ±TRAP) of other promoters tested in the ‘promoter-tbs-GFP’ configuration in co-transfected HEK293T cells±pEF1a-coTRAP[H6] (note that total fold repression is related to promoter strength due to a greater differential between ‘on’ levels compared to untransfected cells). Data are mean average values±s.d. [log10-transformed data] (n=3); *P<0.001 [Welch’s t-test]. (CMVp, cytomegalovirus promoter; EF1a, Elongation factor 1 alpha promoter; INT, synthetic intron; tbs, TRAP-binding sequence; coTRAP[Hisx6], codon-optimized tryptophan RNA-binding attenuation protein with C-terminal Histine tag; polyA, polyadenylation signal; SFFV, spleen focus-forming virus promoter; CMV+betaglb intron; CMV promoter with human beta globin intron; RSV, Rous Sarcoma Virus promoter; HSV-TK, Herpes simplex virus thymidine kinase promoter; SV40, Simian virus 40 promoter; huPGK, human phosphoglycerate kinase 1 promoter). are representative of two independent experiments.
