Journal: Scientific Reports

Article Title: Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide

doi: 10.1038/srep18728

Figure Lengend Snippet: ( A , B ) Normalized time courses (ThT fluorescence data points) for aggregation reactions starting from 0.8 (grey) to 10 μM (red) Aβ42-A2V monomer in 20 mM NaP, 0.2 mM EDTA, 0.02% NaN 3 , pH 8.0. Each colour shows four technical replicates at each concentration. The solid lines represents the best fits of model 3a (A) and model 4a (B) and plots for all models are shown in . ( A ) The best fit with a model consistent with the Aβ42 wt data: primary nucleation, secondary nucleation and elongation with fixed reaction orders n c = n 2 = 2 (as for wt) (model 3a) and ( B ) the best fit with a model including primary nucleation and multi-step secondary nucleation (model 4a). ( C ) The half time as a function of peptide concentration from the best fit of each model is compared to the experimental data. ( D ) Error square sum, a measure of the goodness of the fit, of each model relative to model 4a. The models, with the number of fitting parameters given in brackets, are: Model1b Primary nucleation and elongation (2). Model2b Primary nucleation, fragmentation and elongation (3). Model3a Primary nucleation, secondary nucleation and elongation (2). Model3b Primary nucleation, secondary nucleation and elongation (4). Model4a Primary nucleation, multi-step secondary nucleation and elongation (3). Although involving one less free parameter than model 3b, model 4a yields a lower error. See for the processes and parameters for each model. ( E ) Normalized aggregation kinetics data for samples that initially contain 2.3 μM monomer supplemented with 0.03, 0.1, 0.3, 1, 3, 10 or 30% seeds (in monomer equivalents) confirm the strong role of surface-catalyzed secondary nucleation. The solid lines are fits of model 4a, using the parameter values found above and one free parameter, the elongation rate constant k + . ( F ) Comparison of k 2 k + and k 2 /kn for Aβ42-A2V relative to Aβ42-wt. In particular note the large increase in the relative importance of secondary over primary nucleation.

Article Snippet: A synthetic gene with E. coli optimised codons for Aβ(M1-42) with the A2V mutation cloned into the Pet3a vector was purchased from Genscript (Piscataway; New Jersey, USA).

Techniques: Fluorescence, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: Replication and parMRC locus of pCW3: parM C is shown in yellow, parR C is shown in blue, the parC C site (four direct repeats shown in green) is upstream of parM C , the five inverted repeats (IR) of oriV (IR1 in orange, IR2 in pink, IR3 in lavender, IR4 in blue, IR5 in purple) are shown upstream of rep , which is indicated by the red arrow.

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques:

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: ParR C (pCW3) binds to a cognate parC C (pCW3) sequence. (A) Schematic of the parC C (pCW3) fragment array that consists of 30 bp fragments that overlap by 20 bp, direct repeats are indicated above the fragment array in red. (B) Representative ParR C (pCW3) binding to the parC C (pCW3) fragment array as determined by SPR. (C) Representative SPR binding curves for ParR C (pCW3) and parC C (pCW3) fragments, ParR C (pCW3) + C3 binding curve is shown in blue, and ParR C (pCW3) + C12 binding curve is shown in red. AUC sedimentation velocity experiments were also conducted on ParR C (pCW3), parC C (pCW3) fragment C5 and ParR C (pCW3) and parC C (pCW3) fragment C5 in combination. (D) The continuous sedimentation coefficient distribution [ c ( s )] as a function of normalised sedimentation coefficient ( s 20,W ) for ParR C (pCW3). (E) The continuous mass distribution c ( M ) distribution as a function of molecular mass (Da) for ParR C (pCW3). (F) The continuous sedimentation coefficient distribution [ c ( s )] as a function of s 20,W for parC C (pCW3) C5 (red), ParR C (pCW3) (blue) and, ParR C (pCW3) and parC C (pCW3) C5 in combination (green). Residuals for each fit are shown as insets, confirming the validity of the fit of the data.

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques: Sequencing, Binding Assay, Sedimentation

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: Surface plasmon resonance analysis demonstrated that ParR homologues bind to their cognate parC sites. A) Schematic of parC overlapping fragments. parC B (pJIR4165), parC C (pCW3) and parC D (pJIR3118) fragment arrays were constructed to test binding of ParR homologues to each parC region. All fragment arrays consisted of 30 bp oligonucleotides with 20 bp of overlapping sequence and were designed using POOP. Antisense oligonucleotides were constructed with the ReDCaT linker sequence present at the 3’ end of each fragment in the diagram above. Oligonucleotides were annealed before being captured onto the ReDCaT primed Streptavidin (SA) chip via the complementary base pairing between the ReDCaT linker and the complementary ReDCaT sequence on the Biacore T200 chip. B) SPR profiles obtained when ParR B (pJIR4165) was tested against parC B (pJIR4165) (blue), parC C (pCW3) (Green) and parC D (pJIR3118) (Orange) C) Shows ParR C (pCW3) binding profiles, D) Shows ParR D (pJIR3118) binding profiles. The first lane in every binding graph shows a no protein control with the fragments C1, B1 and D1.

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques: SPR Assay, Construct, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: JGS1987 ParR homologues bind to non-cognate parC from the same family. ParR B , ParR C and ParR D homologues from the C. perfringens isolate, JGS1987, were tested against parC B (pJIR4165), parC C (pCW3) and parC D (pJIR3118) fragment arrays, and binding stability was measured using surface plasmon resonance. A) Shows ParR B (pJGS1987B) binding profiles when used to challenge parC B (pJIR4165) (blue), parC C (pCW3) and parC D (pJIR3118). B) Shows ParR C (pJGS1987C) binding profiles (blue) C) Shows ParR D (pJGS1987D) binding profiles (orange). The first fragment in every graph shows a no protein control

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques: Binding Assay, SPR Assay