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. 2019 May 11;20(9):2343. doi: 10.3390/ijms20092343

Figure 2.

Figure 2

Characterization of ncAA incorporation by ONBYRS-1 into superfolder green fluorescent protein (sfGFP) reporter constructs and comparison with previously reported aaRSs (all expressed with corresponding tRNAs). (A) Schematic illustration of sfGFP reporter constructs for monitoring ncAA incorporation efficiency at one or five positions in sfGFP. Relative positions of the in-frame amber stop codons are indicated (red lines, the sfGFP WT control construct is free of these). (B) SDS-PAGE and anti-Strep Western blot analysis of sfGFP(1TAG) expression. Proteins were produced in E. coli BL21(DE3) using ONBYRS-1 in the presence or absence of 1 mM ONBY as indicated (FL = full-length, TR= truncated product). (C) Fluorescence reporter measurements for the incorporation of ONBY into sfGFP(1TAG) using E. coli BL21(DE3) cells. (D) Fluorescence reporter measurements for the incorporation of ONBY into of sfGFP(5TAG) using E. coli C321.ΔA.exp(DE3) cells. (C,D) Fluorescence values are normalized to strain-specific sfGFP WT fluorescence and to the OD600 of the bacterial culture. In case of ncAA supplementation, 1 mM ONBY was used. Data represent mean ± s.d. of biological triplicates.