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. Author manuscript; available in PMC: 2020 Oct 6.
Published in final edited form as: Nat Chem Biol. 2020 Apr 6;16(5):570–576. doi: 10.1038/s41589-020-0507-z

Fig. 3 |. Simultaneous decoding of two unnatural codons.

Fig. 3 |

a, Schematic illustration of gene cassette containing sfGFP190,200(GXT,AXC), M. mazei tRNAPyl(AYC), and M. jannaschii tRNApAzF(GYT). b-c, Time-course plot of normalized fluorescence during sfGFP expression in the presence of denoted ncAAs (aTc added at t = 0; n = 3, biological replicates; mean and individual data points shown). b, Clonal SSO expression of the cassette in a as well as controls showing expression of cassettes containing only single codons with the appropriate tRNA. c, Clonal expression of a cassette containing sfGFP190,200(TAA,TAG), M. mazei tRNAPyl(TTA), and M. jannaschii tRNApAzF(CTA) also shown, as well as control cassettes containing the single stop-codons with the appropriate suppressor tRNA. d, Pseudocolored western blots (green) and TAMRA fluorescence scans (blue) of purified sfGFP from SSOs in b-c, with and without conjugation to TAMRA-PEG4-DBCO by SPAAC. Images are cropped from the same blots (UBP constructs and stop codon suppressors) but positioned to align the unshifted band in order compare electrophoretic migration. e, Time-course plot of normalized fluorescence during clonal expression of double codon/tRNA cassettes from b-c, with addition of PrK and pAzF. Mean and individual data points shown (n = 3, biological replicates). f, Pseudocolored western blots (green) and TAMRA fluorescence scans (blue) of purified sfGFP from SSOs in e, with and without conjugation to TAMRA-PEG4-DBCO by SPAAC and to TAMRA-PEG4-azide by CuAAC. Uncropped scans of western blots (d, f) shown in Supplementary figure 11.