Journal: Molecular and biochemical parasitology

Article Title: A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

doi: 10.1016/j.molbiopara.2016.08.001

Figure Lengend Snippet: [A] Schematic of the plasmid, showing the 5′ UTR targeting segment (1; 5′ UTR), a segment (3) containing the blasticidin selection marker (BSD), alpha/beta tubulin intergenic rection (INTER), triple-Ty1 tag (3X Ty1), the first 500 bp of FC1 (2; FC1 Cod). and the endogenous tagging vector backbone for inducible expression (ET vector, 4). Sizes for each DNA segment are shown in parentheses. [B] Agarose gel showing the DNA segments used for Gibson assembly (1–4, as labeled in A), and the product (P) of the assembly digested with PacI and NsiI to show the completed endogenous tagging insert. The asterisk in lane 3 denotes the BLA-INTER-3X Ty1 DNA segment, which was excised from a previously-assembled construct by BamHI and HindIII restriction digest. The asterisk in lane 4 denotes the ET plasmid backbone, which was isolated by PacI and NsiI digest. [C] Cells containing the FC1 endogenous tagging construct were fixed and stained with an antibody against the flagella connector (FC; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.

Article Snippet: The homemade Master Mix was prepared by combining 699 μL water, 320 μL 5x isothermal reaction buffer (500 mM Tris-Cl, pH 7.5, 250 mg/mL PEG-8000, 50 mM MgCl 2 , 50 mM DTT, 1 mM each of four dNTPs, 5 mM beta-NAD), 0.64 μL T5 Exonuclease (Epicentre, 10 U/μL), 20 μL Phusion DNA polymerase (NEB, 2 U/μL) and 160 μL Taq DNA ligase (NEB, 40 U/μL).

Techniques: Plasmid Preparation, Selection, Marker, Expressing, Agarose Gel Electrophoresis, Labeling, Construct, Isolation, Staining, Fluorescence, Microscopy

Journal: Molecular and biochemical parasitology

Article Title: A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

doi: 10.1016/j.molbiopara.2016.08.001

Figure Lengend Snippet: [A] Schematic of the plasmid, showing the last 500 bp of the 4400 gene, which functions as a targeting segment (1; 4400 Cod), the triple-Ty1 tag (2; 3X Ty1), the alpha/beta tubulin intergenic region (3; INTER), the puromycin resistance gene (4; PAC), a 500 bp segment of the 3′ UTR of 4400 used for targeting (5; 3′ UTR), and the endogenous tagging vector backbone for inducible expression (ET vector, 6). Sizes for each DNA segment are shown in parentheses. [B] Agarose gel showing the DNA segments used for Gibson assembly (1–6, as labeled in A), and the product (P) of the assembly digested with PacI and NsiI to show the tagged insert. The asterisk in lane 6 denotes the plasmid backbone of the ET vector, which was used for the Gibson reaction. [C] Cells containing the 4400 endogenous tagging construct were fixed and stained with an antibody that detects the basal body and bilobe structure (BB + Bilobe; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.

Article Snippet: The homemade Master Mix was prepared by combining 699 μL water, 320 μL 5x isothermal reaction buffer (500 mM Tris-Cl, pH 7.5, 250 mg/mL PEG-8000, 50 mM MgCl 2 , 50 mM DTT, 1 mM each of four dNTPs, 5 mM beta-NAD), 0.64 μL T5 Exonuclease (Epicentre, 10 U/μL), 20 μL Phusion DNA polymerase (NEB, 2 U/μL) and 160 μL Taq DNA ligase (NEB, 40 U/μL).

Techniques: Plasmid Preparation, Expressing, Agarose Gel Electrophoresis, Labeling, Construct, Staining, Fluorescence, Microscopy