Journal: Frontiers in Molecular Biosciences

Article Title: Advancing Trypanosoma cruzi N-myristoyltransferase as a drug target for Chagas disease through in silico discovery and biochemical evaluation

doi: 10.3389/fmolb.2025.1666768

Figure Lengend Snippet: Cloning strategy and validation of the TcNMT expression construct in the pET-15b vector. (A) Map of the recombinant plasmid pET-15b/TcNMT (7056 bp), showing the insertion of the 1365 bp TcNMT open reading frame (ORF) between NdeI and BamHI restriction sites. Key annotated features include the T7 promoter, lac operator, ribosome binding site (RBS), thrombin cleavage site, 6xHis tag, and the ampicillin resistance gene (AmpR). (B) A linear representation of the construct is shown below the map. (C) PCR amplification of TcNMT from Trypanosoma cruzi CL Brener genomic DNA using an annealing temperature gradient (60 °C, 64 °C, and 68 °C). A clear 1365 bp band corresponding to the expected ORF was observed at all temperatures, with optimal specificity at 64 °C. NC = no-template control. DNA ladder: 100 bp DNA Ladder (Invitrogen, Cat. No. 15628019). (D) Restriction digestion analysis of TOPO: TcNMT and empty TOPO vectors using EcoRI and XbaI. The undigested TOPO: TcNMT plasmid is shown alongside the digested TOPO: TcNMT sample, which released the expected 1,365 bp insert. DNA ladder: E-Gel 1 Kb Plus DNA Ladder (Invitrogen, Cat. No. 10488090). (E) Screening of plasmid DNA extracted from colonies C1–C12 by NdeI and BamHI digestion. Positive colonies displayed two bands corresponding to the TcNMT insert (1,365 bp) and vector backbone. Colony 4 (C4) showed a clear, specific insert band and was selected for further propagation. DNA ladder: E-Gel 1 Kb Plus Express DNA Ladder (Invitrogen, Cat. No. 10488091). (F) Final validation of colony 4 (C4) by BamHI/XbaI digestion prior to sequencing. The digested C4 plasmid shows the expected two-band pattern, including the released 1,365 bp insert and vector backbone. OP = original, undigested pET-15b plasmid used for transformation. The release of the expected insert confirmed proper construction of the expression plasmid. DNA ladder: GeneRuler 1 Kb DNA Ladder (Thermo Fisher Scientific, Cat. No. SM0312).

Article Snippet: Genomic DNA was isolated from CL Brener epimastigotes, and the 1.3 kb TcNMT ORF was amplified using Pfu DNA polymerase (Promega Corporation, Madison, WI, United States) under the following conditions: initial denaturation at 95 °C for 2 min; followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1.5 min; with a final extension at 72 °C for 10 min. PCR and digestion products were analyzed by agarose gel electrophoresis alongside the appropriate DNA size markers, as follows: 100 bp DNA Ladder (Invitrogen, Cat. No. 15628019) for , E-Gel 1 Kb Plus DNA Ladder (Invitrogen, Cat. No. 10488090) for , E-Gel 1 Kb Plus Express DNA Ladder (Invitrogen, Cat. No. 10488091) for , and GeneRuler 1 Kb DNA Ladder (Thermo Fisher Scientific, Cat. No. SM0312) for .

Techniques: Cloning, Biomarker Discovery, Expressing, Construct, Plasmid Preparation, Recombinant, Binding Assay, Amplification, Control, Sequencing, Transformation Assay