Fig 1. Engineering the UAA system and demonstrating efficient incorporation of Azi in T. gondii.

(A) Diagram showing the use of amber stop codon suppression to incorporate UAAs into a nascent peptide strand using the endogenous translation machinery. (B) Chemical structure of the photoreactive UAA p-azidophenylalanine (Azi). Exposure of Azi to UV-A (365-nm) ultraviolet light causes the azide group to irreversibly form a reactive nitrene intermediate, which forms covalent crosslinks with proximal proteins. (C) Construct showing the Ty1-tagged aminoacyl-tRNA synthetase (E2AziRS) driven by the constitutively active GRA1 promoter and three tandem cassettes of the cognate amber suppressor tRNA driven by the RNA polymerase III–specific U6 promoter. (D) IFA showing that stably expressed E2AziRS localizes to the parasite cytoplasm as expected. Green, mouse anti-Ty1. Scale bar represents 2 μm. (E) Construct showing the SAG1 gene driven by the GRA1 promoter, in which the second amino acid F2 has been mutated to an amber codon. (F) IFA showing RHΔhxgprtΔsag1 parasites stably transfected with the synthetase/tRNA and SAG1 constructs. E2AziRS expression is confirmed by anti-Ty1 staining. Without Azi in the growth medium, SAG1 is not detected due to the in-frame stop codon. Upon addition of Azi, robust expression of SAG1 is observed trafficking properly to the cell periphery. Red, rabbit anti-SAG1 antibody; green, mouse anti-Ty1 antibody. Scale bar represents 2 μm. (G) Western blot of whole cell lysates shows expression of the SAG1 nonsense mutant only when Azi is added to the medium. Wild-type RH parasites expressing endogenous SAG1 are used as a control. Azi, p-azidophenylalanine; E2AziRS, Azi-tRNA synthetase; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyl transferase; IFA, immunofluorescence assay; SAG1, surface antigen 1; UAA, unnatural amino acid; WT, wild-type.