Fig 2. Selection for in-frame TAG replacement.

(A) Schematic of the cytochrome (red) and sfGFP (green) fusion with the TEV cleavage site between the two shown. The residue targeted (K51) for replacement and the rational TAG replacements are shown. (B) cellular fluorescence of each variant in the absence (-nAA) and presence (+nAA) of either azF or iodoF. The wt refers to the cyt b ₅₆₂-sfGFP fusion without any TAG codon replacement. (C) SDS-PAGE (top panel) and Western blot (bottom panel) analysis of fusion protein expression in the soluble fraction of the cell lysate. The—v lane represents cells producing CG-wt in the absence of the pAB vector. The + and—lanes refer to the presence or absence of the nAA, respectively. CG and G refer to the cyt b ₅₆₂-sfGFP fusion (~40 kDa) and the sfGFP alone (27 kDa) generated through TEV cleavage. The Western blot detection was performed with an antiGFP primary antibody.