Figure 1.
Comparison of the tandem copy and H1 promoter plasmid designs.
The six-tandem approach (left) was replaced with a plasmid containing a human H1 promoter (right). A 3′ flanking sequence was also used to terminate transcription. The tRNA gene sequences are identical between the two plasmid designs, except in the 3′ CCA tail. The construct on the left contains this sequence while that on the right does not (EZ Plasmid Map).
Figure 2.
Acceptor stem sequences of the various tRNA constructs.
Each tRNA construct was cloned into a plasmid, downstream of the human H1 promoter. Wt-tRNACUA is identical to M. jannaschii tyrosyl-tRNA, except in the anticodon region, where it contains a C35 (pknotsRG, BiBiServ).
Figure 3.
Assessing the orthogonality of various tRNA constructs in HEK293T cells.
Full-length GFP was visualized by UV-light 72 hours after transfection. Expression of full-length GFP will only occur in the presence of amino acid charged suppressor tRNA. Since no exogenous aaRS was provided, full-length GFP expression implies that endogenous aaRS was able to recognize and charge the tRNA with an amino acid. Cells were transfected with the following plasmids: (A) p-EGFP-N1 (B) p-GFP_39TAG (C) p-GFP_39TAG and H1_wt-tRNACUA (D) p-GFP_39TAG and H1_1bp-tRNACUA (E) p-GFP_39TAG and H1_2bp-tRNACUA (F) p-GFP_39TAG and H1_3bp-tRNACUA.
Table 1.
Assessing the orthogonality of various tRNA constructs in HEK293T cells.
Figure 4.
Alignment of the amino acid sequences of the CP1-transplanted mutants.
Inserted CP1 sequences are shown in black. Six sequences used were from E. coli TyrRS, while two were taken from T. thermophilus TyrRS. Both bacterial TyrRSs recognize a G1:C72 containing tyrosyl-tRNA. Each CP1 swapped TyrRS was tested for the ability to charge 1bp-tRNACUA in HEK293T cells. (Bioworkbench, SDSC).
Table 2.
Ratio of the suppression efficiency to tRNA background.