The full coding region of IMC29 was PCR amplified from cDNA using primers P21/P22 and cloned into a UPRT-locus knockout vector (10 (link)) using BglII/NotI (all enzymes purchased from NEB). The endogenous promoter was amplified from genomic DNA using primers P23/P24 and inserted with NsiI/BglII upstream of the coding sequence, resulting in UPRTKO-IMC29pro-IMC29FL. This complement vector was then linearized with DraIII-HF and transfected into Δimc29PC parasites along with a pU6 that targets the UPRT coding region. Selection was performed with 5 μg/mL 5-fluorodeoxyuridine (FUDR) for replacement of UPRT (57 (link)). Potential clones were screened by IFA, and an HA-positive clone was designated IMC29FL.
For IMC29 deletion constructs, UPRTKO-IMC29pro-IMC29FL was used as the template to amplify truncations from the IMC29 coding region, using primers P25 to P31. The P22 reverse primer was utilized to amplify each N-terminal truncation. The P21 forward primer was utilized to amplify the two C-terminal truncations. Each insert was cloned into a BglII/NotI-digested UPRTKO-IMC29pro-IMC29FL. For the additional C-terminal truncation (Δ1111-1218), the UPRTKO-IMC29pro-IMC29FL plasmid was used as the template using the Q5 mutagenesis kit. Primers P36 and P37 were used for inverse PCR to amplify the entire plasmid except for residues 1111 to 1218. For the phosphorylation mutant construct, phosphorylation sites were annotated from ToxoDB, combining phosphoproteomic data of both TgME49_243200 and TgGT1_243200. These include T42, S47, S50, S52, S68, S69, S73, T80, S205, S206, S526, S528, S736, T778, T779, T795, S797, T811, S813, S815, S817, S844, S846, T977, S1087, T1090, T1097, Y1100, S1105, S1106, T1220, and T1230. The mutated residues were designed in three synthetic gene blocks and ligated together using BglII/SgrAI, SgrAI/BamHI, and BamHI/NotI to generate a full-length IMC29 with all 32 residues mutated simultaneously to alanine. The same processes for linearization, transfection, and selection were followed for all deletion and mutant constructs.
For IMC29 deletion constructs, UPRTKO-IMC29pro-IMC29FL was used as the template to amplify truncations from the IMC29 coding region, using primers P25 to P31. The P22 reverse primer was utilized to amplify each N-terminal truncation. The P21 forward primer was utilized to amplify the two C-terminal truncations. Each insert was cloned into a BglII/NotI-digested UPRTKO-IMC29pro-IMC29FL. For the additional C-terminal truncation (Δ1111-1218), the UPRTKO-IMC29pro-IMC29FL plasmid was used as the template using the Q5 mutagenesis kit. Primers P36 and P37 were used for inverse PCR to amplify the entire plasmid except for residues 1111 to 1218. For the phosphorylation mutant construct, phosphorylation sites were annotated from ToxoDB, combining phosphoproteomic data of both TgME49_243200 and TgGT1_243200. These include T42, S47, S50, S52, S68, S69, S73, T80, S205, S206, S526, S528, S736, T778, T779, T795, S797, T811, S813, S815, S817, S844, S846, T977, S1087, T1090, T1097, Y1100, S1105, S1106, T1220, and T1230. The mutated residues were designed in three synthetic gene blocks and ligated together using BglII/SgrAI, SgrAI/BamHI, and BamHI/NotI to generate a full-length IMC29 with all 32 residues mutated simultaneously to alanine. The same processes for linearization, transfection, and selection were followed for all deletion and mutant constructs.
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