ptarget  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons

doi: 10.1093/nar/gkaf149

Figure Lengend Snippet: PmcCAST TnsC forms a heptameric complex in the presence of ATP and DNA. ( A ) Schematic representation of the type I-B CAST system from Peltigera membranacea cyanobiont 210A . LE and RE denote left and right transposon ends. ( B ) Size-exclusion chromatography profiles of TnsC alone, in the presence of ATP, double-stranded (ds)DNA, or both ATP and dsDNA. ( C ) Representative negative-stain electron micrographs of TnsC in the presence or absence of dsDNA and/or ATP. Scale bar corresponds to 100 nm. ( D ) Single-particle cryo-EM reconstruction of the dsDNA- and AMPPNP-bound TnsC heptamer (from left to right: top, side and bottom views). ( E ) Structural model of the dsDNA- and AMPPNP-bound TnsC heptamer (from left to right: top, side and bottom views). Individual TnsC protomers are labeled with distinct superscript letters to differentiate each subunit. AMPPNP molecules are shown in space-fill representation.

Article Snippet: To remove the tRNA gene from the pTarget vector, a pTarget_ΔtRNA vector was created by Gibson assembly between two fragments amplified from the template pTarget (PmcCAST) (Addgene #168163) using the primers pTarget_ΔtRNA_1_f with pTarget_ΔtRNA_1_r, and pTarget_ΔtRNA_2_f with pTarget_ΔtRNA_2_r ( ).

Techniques: Size-exclusion Chromatography, Staining, Single Particle, Cryo-EM Sample Prep, Labeling

Journal: Nucleic Acids Research

Article Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons

doi: 10.1093/nar/gkaf149

Figure Lengend Snippet: TnsC oligomerization, DNA binding and ATP hydrolysis are required for transposition. ( A ) Inter-protomer interfaces within the TnsC heptamer. Regions depicted in detail in subsequent panels are indicated with dashed boxes. ( B ) Detailed view of the ATPase catalytic pocket. Bound AMPPNP and interacting amino acid residues are shown in stick format. Mg 2+ ion is depicted as a green sphere. ( C ) RNA-guided transposition activity in E. coli of PmcCAST systems containing mutations in the catalytic pocket of TnsC, as quantified by ddPCR. Data are presented as mean ± s.d. of three biologically independent replicates ( n = 3), each measured in technical duplicates. Bars labeled as n.d. (“non-detectable”) indicate values below the detection limit of the assay. The positive control (“CTRL+”) corresponds to a ddPCR reaction that uses as DNA template an artificial pTarget plasmid with the transposon DNA from the pDonor plasmid inserted at the target site in the left end–right end (LE–RE) orientation. ( D – F ) Detailed views of the TnsC inter-protomer interfaces. ( G ) RNA-guided transposition efficiency in E. coli of PmcCAST systems containing mutations in the inter-protomer interfaces, quantified by ddPCR as in panel (C). ( H ) Detailed view of the DNA-binding interfaces. Only residues within 3.5 Å of the DNA are displayed. ( I ) RNA-guided transposition efficiency in E. coli of PmcCAST systems containing mutations in the DNA-binding interfaces, quantified by ddPCR as in panel (C).

Article Snippet: To remove the tRNA gene from the pTarget vector, a pTarget_ΔtRNA vector was created by Gibson assembly between two fragments amplified from the template pTarget (PmcCAST) (Addgene #168163) using the primers pTarget_ΔtRNA_1_f with pTarget_ΔtRNA_1_r, and pTarget_ΔtRNA_2_f with pTarget_ΔtRNA_2_r ( ).

Techniques: Binding Assay, Activity Assay, Labeling, Positive Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons

doi: 10.1093/nar/gkaf149

Figure Lengend Snippet: The C-terminal hook in TnsAB interacts with heptameric TnsC. ( A ) Schematic diagram of the domain organization of type I-B PmcCAST TnsAB. ( B ) Coprecipitation of TnsC by amylose-immobilized TnsAB (full-length or C-terminal domain), fused to MBP) in the presence of different nucleotides (ADP, AMPPNP, ATP; bottom). I: 10% input; E: elution; C: TnsC-only control; C + AB: TnsC and MBP-TnsAB full-length sample; C + AB CTD : TnsC and MBP-TnsAB C-terminal domain sample. ( C ) Single-particle cryo-EM reconstruction of the heptameric TnsC–DNA–AMPPNP complex bound to six copies of the TnsAB hook (top and side views). Individual TnsC subunits are indicated with different colours. The TnsAB hooks densities are depicted in bright yellow. ( D ) Structural model of the TnsC–DNA–AMPPNP complex bound to six copies of the TnsAB hook (top and side views). Individual TnsC protomers are labeled with distinct superscript letters to differentiate each subunit. The TnsAB hooks are depicted in space-fill representation (bright yellow). ( E ) Detailed view of the TnsAB hook and the TnsC interface. ( F ) RNA-guided transposition activity in E. coli of PmcCAST systems containing mutations in the TnsAB hook and TnsC interface, as quantified by ddPCR. Data are presented as mean ± s.d. of three biologically independent replicates ( n = 3), each measured in technical duplicates. Bars labeled n.d. (“non-detectable”) indicate values below the detection limit of the assay. The positive control (“CTRL+”) corresponds to a ddPCR reaction that uses as DNA template an artificial pTarget plasmid with the transposon DNA from the pDonor plasmid inserted at the target site in the left end-right end (LE–RE) orientation.

Article Snippet: To remove the tRNA gene from the pTarget vector, a pTarget_ΔtRNA vector was created by Gibson assembly between two fragments amplified from the template pTarget (PmcCAST) (Addgene #168163) using the primers pTarget_ΔtRNA_1_f with pTarget_ΔtRNA_1_r, and pTarget_ΔtRNA_2_f with pTarget_ΔtRNA_2_r ( ).

Techniques: Control, Single Particle, Cryo-EM Sample Prep, Labeling, Activity Assay, Positive Control, Plasmid Preparation