Journal: Digestive Diseases and Sciences

Article Title: Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

doi: 10.1007/s10620-016-4422-9

Figure Lengend Snippet: IL-37 and CXCL8 protein expressions in response to 10 or 100 ng/ml flagellin stimulation of T84 cells for different time periods. Protein levels of intracellular IL-37 ( a ), secreted IL-37 ( b ), and secreted CXCL8 ( c ) in cell culture media were compared to unstimulated controls ( n = 4). Statistically significant differences are marked as *, **, or *** and depict p < 0.05, < 0.01, or < 0.001, respectively. All data are shown as mean ± SEM

Article Snippet: To detect the optimal concentration of TLR5 stimulation with its ligand flagellin, recombinant flagellin from Salmonella typhimurium (Novus Biologicals, Cambridge, UK) was used [ ].

Techniques: Cell Culture

Journal: Digestive Diseases and Sciences

Article Title: Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

doi: 10.1007/s10620-016-4422-9

Figure Lengend Snippet: mRNA ( a ) and protein ( b ) levels of CCL2, CCL5, CXCL8, CXCL10, and CXCL11 ( n = 5). The filled bars represent cells stimulated with 100 ng/ml flagellin for 12 h, whereas empty bars denote unstimulated cells. First, unstimulated cells with reduced IL-37 levels (IL-37sgRNA) were compared to unstimulated cells transfected with an empty plasmid (TFneg), whereas the IL-37sgRNA cells stimulated with flagellin are compared to TFneg cells stimulated with flagellin. Second, unstimulated cells are compared to flagellin-stimulated cells, within the same cell type (WT, TFneg or IL-37sgRNA). Statistically significant differences are marked with *, **, or *** depicting p < 0.05, < 0.01, or < 0.001. All data are shown as mean ± SEM. WT wild-type T84 cells, N.D not detected

Article Snippet: To detect the optimal concentration of TLR5 stimulation with its ligand flagellin, recombinant flagellin from Salmonella typhimurium (Novus Biologicals, Cambridge, UK) was used [ ].

Techniques: Transfection, Plasmid Preparation

Journal: Digestive Diseases and Sciences

Article Title: Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

doi: 10.1007/s10620-016-4422-9

Figure Lengend Snippet: mRNA ( a ) and protein ( b ) levels of CCL3, CX 3 CL1, and CCL20 ( n = 5). The filled bars represent cells stimulated with 100 ng/ml flagellin for 12 h, whereas empty bars denote unstimulated cells. First, unstimulated cells with reduced IL-37 levels (IL-37sgRNA) were compared to unstimulated cells transfected with an empty plasmid (TFneg), whereas the IL-37sgRNA cells stimulated with flagellin are compared to TFneg cells stimulated with flagellin. Second, unstimulated cells are compared to flagellin-stimulated cells, within the same cell type (WT, TFneg or IL-37sgRNA). Statistically significant differences are marked with *, **, or *** depicting p < 0.05, < 0.01, or < 0.001. All data are shown as mean ± SEM. WT wild-type T84 cells, N.D not detected

Article Snippet: To detect the optimal concentration of TLR5 stimulation with its ligand flagellin, recombinant flagellin from Salmonella typhimurium (Novus Biologicals, Cambridge, UK) was used [ ].

Techniques: Transfection, Plasmid Preparation

Journal: Digestive Diseases and Sciences

Article Title: Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

doi: 10.1007/s10620-016-4422-9

Figure Lengend Snippet: mRNA ( a ) and protein ( b ) levels of CCL4, CCL22, and CXCL9 ( n = 5). The filled bars represent cells stimulated with 100 ng/ml flagellin for 12 h, whereas empty bars denote unstimulated cells. First, unstimulated cells with reduced IL-37 levels (IL-37sgRNA) were compared to unstimulated cells transfected with an empty plasmid (TFneg), whereas the IL-37sgRNA cells stimulated with flagellin are compared to TFneg cells stimulated with flagellin. Second, unstimulated cells are compared to flagellin-stimulated cells, within the same cell type (WT, TFneg or IL-37sgRNA). Statistically significant differences are marked with *, **, or *** depicting p < 0.05, < 0.01, or < 0.001. All data are shown as mean ± SEM. WT wild-type T84 cells, N.D not detected

Article Snippet: To detect the optimal concentration of TLR5 stimulation with its ligand flagellin, recombinant flagellin from Salmonella typhimurium (Novus Biologicals, Cambridge, UK) was used [ ].

Techniques: Transfection, Plasmid Preparation

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 1 | Culture supernatants from Salmonella typhimurium promotes NLRC4-dependent inflammasome activation. (A) Illustration of experimental scheme to determine inflammasome activation in BMDMs by bacteria-secreted molecules using transwell plate. (B) Immunoblots from mouse BMDMs in the lower well (A) incubated with wild-type (WT) or DfliC-fljB S. typhimurium SL1344 in the upper well (A, MOI 30) for 7 h. (C) Immunoblots from mouse BMDMs untreated or treated with culture supernatant (CS, 1/33 or 1/10 volume of culture medium), derived from 6 h culture of WT or DfliC–fljB S. typhimurium, for 6 h. (D) Immunoblots from Nlrc4+/+ or Nlrc4−/−mice BMDMs treated with WT or DfliC–fljB S. typhimurium CS (1/10) for 6 h. (E) Immunoblots from Nlrp3+/+ or Nlrp3−/−mice BMDMs treated with WT or DfliC–fljB S. typhimurium CS (1/10) for 6 h, or primed with LPS (0.25 µg/ml, 3 h), followed by treatment with ATP (2.5 mM, 40 min). (F) Immunoblots from mouse BMDMs incubated with S. typhimurium CS (1/20) with or without heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min), for 6 h. (G, H) Quantification of IL-1b (G) or IL-6 (H) in the supernatant of mouse BMDMs treated with S. typhimurium CS (1/20) as same as in (F). (n = 3, one-way ANOVA) (I, J) Quantification of IL-1b (I) or IL-6 (J) in the culture supernatants of mouse BMDMs treated with WT, DfliC–fljB, or DfliC–fljB–prgJ S. typhimurium CS (1/100) for 6 h. (n = 4, one-way ANOVA). (K) Immunoblots from mouse BMDMs treated with culture supernatant (CS, 1/100 or 1/20), derived from 6 h culture of WT, DfliC–fljB, DfliC–fljB–prgJ S. typhimurium 14028s, for 6 h. (L) Immunoblots from mouse BMDMs treated with S. typhimurium flagellin (ST-FLA, 250 ng/ml) with or without premixing with DOTAP (DT) liposomal transfection reagent for 6 h or treated with LPS (0.25 µg/ml, 3 h), followed by ATP treatment (2.5 mM, 40 min). Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with S. typhimurium CS-treated group. (*P < 0.05, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Activation Assay, Bacteria, Western Blot, Incubation, Derivative Assay, Transfection

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 5 | Salmonella-released outer membrane vesicles exhibit heat-resistant and GBP2-independent inflammasome activation potential. (A) Immunoblots of OptiPrep density gradient fractions of S. typhimurium OMVs. (B) Immunoblots of the extracts of OMVs in the presence of heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min) by anti-ST-flagellin antibody. (C) Immunoblots from mouse BMDMs primed with LPS (0.25 µg/ml, 3 h), followed by the treatment with intact or heat-treated S. typhimurium flagellin (250 ng/ml) for 6 h. (D) Immunoblots of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs (5 µg/ml) for 6 h. (E) Quantification of IL-1b in the culture supernatants of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs for 6 h. (n = 5). (F) Immunoblots from Gbp2+/+ or Gbp2−/−mice BMDMs treated with E. coli BL21 OMVs (5 µg/ml) or S. typhimurium (14028s) OMVs (5 µg/ml) or for 8 h. (G, H) Quantification of IL-1b in the culture supernatants of Gbp2+/+ or Gbp2−/−mice BMDMs treated as same as (F) (n = 3). (I, J) Immunoblots of mouse BMDMs treated with intact or proteinase-treated S. typhimurium OMVs (10 µg/ml) in the presence of MCC 950 (100 nM) as indicated for 8 h. After proteinase treatment, PMSF was added to eliminate proteinase K activity. Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences. (**P < 0.01, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Membrane, Activation Assay, Western Blot, Activity Assay

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 7 | Proposed mechanism of host inflammasome activation by flagellated or non-flagellated bacteria-released outer membrane vesicles. Flagellated bacteria such as S. typhimurium or P. aeruginosa facilitate the delivery of bacterial flagellin and LPS into the cytoplasm of host cells via OMV cargo. In the initial phase of OMV exposure, OMV-delivered flagellin causes the assembly of NLRC4 inflammasome leading to caspase-1 activation and the subsequent IL-1b secretion. Besides flagellin, intracellular OMV-associated LPS can activate caspase-11 non-canonical inflammasome signaling, which includes GSDMD-dependent pyroptosis and NLRP3-mediated caspase-1 activation and IL-1b secretion. On the contrary, OMVs derived from non-flagellated bacteria such as E. coli mediates the cytosolic delivery of bacterial LPS. Non-flagellated bacterial -delivered LPS mainly causes the activation of caspase-11 and GSDMD-mediated pyroptosis, contributing to potential endotoxic shock. NAIP5, NLR family, apoptosis inhibitory protein 5; NLRC4, NLR family, CARD domain-containing protein 4; NLRP3, NLR family, pyrin domain-containing protein 3; GSDMD, gasdermin D.

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Activation Assay, Bacteria, Membrane, Derivative Assay

Journal: Virology

Article Title: The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting

doi: 10.1016/j.virol.2017.01.023

Figure Lengend Snippet: Schematic representation of epitope tagged ORFs of full length and truncated GPCMV IE1 GFP- and FLAG-tagged expression constructs. All expression constructs are under the control of the immediate early CMV promoter (Pro) and contain a SV40 polyadenylation signal (polyA). Nuclear localization signal (NLS, grey box). Protein expression confirmed by western blot analysis and immunofluorescence. A–C, transient expression and localization of full-length GFP-IE1. D–F, transient expression and localization of GFP-IE1Cdel. G–I, transient expression and localization of FLAG-IE1Ndel. J–L, transient expression and localization of FLAG-IE1NCdel. M–O, transient expression and localization of GFP-IET. FLAG-IE1Ndel and FLAG-IE1NCdel detected by primary anti-epitope antibody and secondary anti-mouse IgG-FITC (immunofluorescence) or anti-mouse IgG-HRP (western blot). GFP-IE1, GFP-IE1Cdel, and GFP-IET were detected by fluorescence and specific epitope antibody (western blot). Lane 1 in A, D, G, J, and M are individual transfected constructs also visualized in B, E, H, K, and N. Lane 2 are mock transfected GPL cells. Panels C, F, I, L, and O are DAPI overlays.

Article Snippet: For immunofluorescence assays, primary antibodies were used at 1/500: mouse anti-HSV-1 ICP0 (Santa Cruz); rabbit anti-HA (Novus Biologicals); mouse anti-FLAG (Novus Biological); mouse anti-GFP (Santa Cruz); mouse anti-Myc-c (Novus Biologicals); rabbit anti-GPCMV IE2.

Techniques: Expressing, Construct, Western Blot, Immunofluorescence, Fluorescence, Transfection

Journal: Virology

Article Title: The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting

doi: 10.1016/j.virol.2017.01.023

Figure Lengend Snippet: (i) Schematic representation of GFP- and FLAG-tagged IE2 fusion protein expression constructs. Expression constructs are under the control of the CMV immediate early promoter (Pro) and contain a SV40 polyadenylation signal (polyA). Nuclear localization signal (NLS, grey box). (ii) A–C, transient expression and localization of GFP-IE2. Lane 1, individually transfected GFP-IE2 visualized in B. Lane 2, mock transfected cell lysate. D–F, transient expression and localization of FLAG-IE2. Lane 1, individually transfected FLAG-IE2 visualized in E. Lane 2, mock transfected cell lysate. (iii) G–I, co-localization of FLAG-IE1 and GFP-IE2. G and H represent individually transfected GFP-IE2 (G) and FLAG-IE1 (H) respectively. I is the merge of G and H overlaid with DAPI. J–L, co-localization of GFP-IE1 (J) and FLAG-IE2 (K). L represents the merge of J and K overlaid with DAPI. (iv) M–O, FLAG-IE1 (N) in the presence of GPCMV (M). O, merge of M and N overlaid with DAPI. P–R, GFP-IE2 (Q) in the presence of GPCMV (P). R, overlay with DAPI. (v) S–U, FLAG-IE2 (T) in the presence of GPCMV (S). U is overlay of S and T. (vi) V–X, GPCMV IE2 (W). X, overlay with DAPI. FLAG-IE1 and FLAG-IE2 detected by primary anti-FLAG Ab and secondary anti-mouse IgG-TRITC (immunofluorescence) or anti-mouse IgG-HRP (western blot). GFP-IE1 and GFP-IE2 were detected by fluorescence and specific epitope Ab (western blot). GPCMV IE2 was detected via IE2-specific antisera. Arrows indicate accumulation of IE2.

Article Snippet: For immunofluorescence assays, primary antibodies were used at 1/500: mouse anti-HSV-1 ICP0 (Santa Cruz); rabbit anti-HA (Novus Biologicals); mouse anti-FLAG (Novus Biological); mouse anti-GFP (Santa Cruz); mouse anti-Myc-c (Novus Biologicals); rabbit anti-GPCMV IE2.

Techniques: Expressing, Construct, Transfection, Immunofluorescence, Western Blot, Fluorescence

Journal: Virology

Article Title: The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting

doi: 10.1016/j.virol.2017.01.023

Figure Lengend Snippet: (i) Co-localization of different IE1 truncation mutants and gpPMLmyc in GPL cells analyzed by immunofluorescence. A–D, GFP-IE1Cdel (A) and gpPMLmyc (B) shown separately in the same cell. C merged image for A and B. D overlay of C with DAPI. E–H, FLAG-IE1Ndel (E) and gpPMLmyc (F). G merged image, with H being DAPI overlay. I–L, FLAG-IE1NCdel (I) and gpPMLmyc (J). K merged image, with L being DAPI overlay. M–P, GFP-IET (M) and gpPMLmyc (N). O merged image, with P being DAPI overlay. (ii) GFP-IE1Cdel and gpPMLmyc co-expression and IP. (iii) GFP-IET and gpPMLmyc co-expression and IP. Lanes 1 and 4, total cell lysate of transfected GPL cells. Lanes 3 and 6, IP reactions. Lanes 2 and 5, mock transfected GPL cells. gpPMLmyc, FLAG-IENdel, and FLAG-IE1NCdel detected by primary anti-epitope Ab and secondary anti-mouse IgG FITC or TRITC (immunofluorescence) and anti-mouse IgG-HRP (western blot). GFP-IE1Cdel and GFP-IET detected by fluorescence (cell localization) and specific epitope Ab (western blot).

Article Snippet: For immunofluorescence assays, primary antibodies were used at 1/500: mouse anti-HSV-1 ICP0 (Santa Cruz); rabbit anti-HA (Novus Biologicals); mouse anti-FLAG (Novus Biological); mouse anti-GFP (Santa Cruz); mouse anti-Myc-c (Novus Biologicals); rabbit anti-GPCMV IE2.

Techniques: Immunofluorescence, Expressing, Transfection, Western Blot, Fluorescence

Journal: Nucleic Acids Research

Article Title: The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity

doi: 10.1093/nar/gkad017

Figure Lengend Snippet: Structural model of IEE from Escherichia coli O157 reference EDL933 strain. The N-terminal AEP domain is colored in cyan and shows the predicted catalytic aspartates responsible for the polymerization reaction (represented as spheres). The C-terminal helicase domain is colored in brown. The conserved lysine that would bind the ATP molecule and the arginine finger are represented as spheres. The predicted structure of IEE from E. coli O157:H7 was obtained through the Uniprot web site ( https://www.uniprot.org/ ) from the Alphafold Database ID A0A0H3JF09 , and used under CC BY4.0. The original colors were changed as described above. Figure was generated using The Open-Source Pymol Molecular Graphics System, v. 2.5.0, Schrödinger, LLC (Open-Source PyMOL is Copyright (C) Schrodinger, LLC.)

Article Snippet: Recombinant IEE : The open reading frame encoding IEE from E. coli O157:H7 reference strain EDL633 [GeneBank Accession number AAG55274.1; ( )] was synthesized by the Cusabio Technology LLC gene synthesis service, and was cloned between the NdeI and BamHI restriction sites in the pET28a(+) vector to express a recombinant protein fused to an N-terminal (His) 6 -tag for purification on Ni +2 -affinity resin.

Techniques: Generated

Journal: Nucleic Acids Research

Article Title: The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity

doi: 10.1093/nar/gkad017

Figure Lengend Snippet: Catalytic active sites of IEE. ( A ) Modular organization of several AEP domain-containing enzymes . The conserved AEP domain (green bar) contains the three conserved regions A, B and C, which form the polymerization active site (in the alignment metal ligands are indicated in red letters and the conserved His that interacts with the incoming nucleotide is in blue). Members such as bacterial NHEJ Ligases D from M. tuberculosis and P. aeruginosa ( Mtu - and Pae LigD) contain additional phosphoesterase (in grey) and ligase (in orange) domains that process 3’-ends and seal the resulting nicks after synapsis during the end-joining reaction. Some members such as the PrimPol of plasmid pRN1 from Sulfolobus islandicus and human PrimPol contain a Zn-finger domain (in magenta), which is essential to stabilize the initiating nucleotide during the DNA priming reaction . The N-terminal AEP domain of IEE is fused to a C-terminal helicase-like domain containing the seven conserved motifs present in superfamily II of these proteins. The figure also shows an alignment of the C-terminal helicase domain of IEE with the superfamily II helicase member RadD from E. coli . Mtu PolDom, Polymerization domain (AEP domain) of Mtu LigD; Pae PolDom, Polymerization domain of Pae LigD; Msm PolD, M. smegmatis PolD1 [PrimPolC ]; pRN1, plasmid pRN1 from S. islandicus ; hPrimPol, human PrimPol. ( B ) Model of the active site geometry of the AEP domain of IEE . The model was obtained by superposition of the putative catalytic residues of the AEP domain of IEE with the crystallographic structure of the ternary complex of PrimPolC [PDB 6SA0 ]. (C) Detailed view of the predicted structure of the C-terminal helicase domain of IEE . The predicted ATP ligand residue Lys451 and the arginine finger (Arg735) are shown as spheres. The location in the three-dimensional structure of the conserved motifs is indicated following the same color code as in the alignment shown in (A). Figures (B) and (C) were generated using The Open-Source Pymol Molecular Graphics System, v. 2.5.0, Schrödinger, LLC (Open-Source PyMOL is Copyright (C) Schrodinger, LLC.)

Article Snippet: Recombinant IEE : The open reading frame encoding IEE from E. coli O157:H7 reference strain EDL633 [GeneBank Accession number AAG55274.1; ( )] was synthesized by the Cusabio Technology LLC gene synthesis service, and was cloned between the NdeI and BamHI restriction sites in the pET28a(+) vector to express a recombinant protein fused to an N-terminal (His) 6 -tag for purification on Ni +2 -affinity resin.

Techniques: Plasmid Preparation, Residue, Generated

Journal: Nucleic Acids Research

Article Title: The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity

doi: 10.1093/nar/gkad017

Figure Lengend Snippet: High abundance of IEE across diverse strains of E. coli . ( A ) Correlation between the most common predicted serotypes (left axis) and multilocus sequence typing (MLST) groups (right axis) among the IEE positive assemblies in GenBank. The serotypes are represented as colored bars and MLST groups as points. ( B ) Phylogenetic groups of the IEE-harboring assemblies. The percentage of each Clermont's classification phylogroup is indicated in the pie sections and the scale in the outer ring represents the actual number of assemblies. ‘Other’ stands for assemblies from minority groups, cryptic phylogroup, and labeled as non- E. coli genomes. See Materials and Methods for details.

Article Snippet: Recombinant IEE : The open reading frame encoding IEE from E. coli O157:H7 reference strain EDL633 [GeneBank Accession number AAG55274.1; ( )] was synthesized by the Cusabio Technology LLC gene synthesis service, and was cloned between the NdeI and BamHI restriction sites in the pET28a(+) vector to express a recombinant protein fused to an N-terminal (His) 6 -tag for purification on Ni +2 -affinity resin.

Techniques: Sequencing, Labeling

Journal: Nucleic Acids Research

Article Title: The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity

doi: 10.1093/nar/gkad017

Figure Lengend Snippet: IEE promotes MMEJ in vitro . ( A ) MMEJ reactions with dsDNA substrates . The assay was performed as described in Materials and Methods, in the presence of 25 nM of the indicated 5’-labeled dsDNA schematized on top of the figure, 40 nM of IEE, and 10 μM of either dNTPs or NTPs, as indicated. After incubation for 15 min at 37°C, the reactions were stopped by adding EDTA up to 10 mM. Position of the substrates is indicated. Bar chart shows the percentage of elongated primers obtained in the presence of either dNTPs or NTPs ( n = 2 each; means ± SEM). ( B ) IEE elongates the protruding 3’-end using the opposing overhang as a template in trans . Assays were performed as described in (A) in the presence of either 25 nM of 5’-labeled dsDNA-4 substrate (left panel) or 1 nM of 5’-labeled dsDNA-4 and 20 nM of the indicated non-labeled dsDNA molecule (right panel). Position of the substrate and the elongation products is indicated. ( C ) Complete MMEJ reaction mediated by IEE and LigA . The assay was performed as described in Materials and Methods, in the presence of 25 nM of dsDNA-6, 40 nM IEE, 10 μM dNTPs, 1 mM MnCl 2 , 5 mM MgCl 2 and, when indicated, 500 μM NAD + and 1 unit of E. coli LigA (NEB). After incubation for 15 min at 37°C, samples were processed and analyzed as described in (A). Position of the substrate and reaction products is indicated.

Article Snippet: Recombinant IEE : The open reading frame encoding IEE from E. coli O157:H7 reference strain EDL633 [GeneBank Accession number AAG55274.1; ( )] was synthesized by the Cusabio Technology LLC gene synthesis service, and was cloned between the NdeI and BamHI restriction sites in the pET28a(+) vector to express a recombinant protein fused to an N-terminal (His) 6 -tag for purification on Ni +2 -affinity resin.

Techniques: In Vitro, Labeling, Incubation