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  • 99
    Millipore phage dna extraction
    Identification of a tranducing phage particle, φSaBov LUK , harboring linear phage <t>DNA.</t> (A) A schematic map of linear phage DNA, based on <t>PCR</t> results (see below). Coloring of genes is as in Fig. 1 . (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoR I restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1 ).
    Phage Dna Extraction, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore phage dna
    mobE and nrdA are co-transcribed. ( A ) Schematic of the nrdA and mobE genes of phage T4, with promoters and putative RNA hairpin sequences indicated. The position of primers used for RT-PCR or northern blot analyses are indicated by arrows, and the length of the products indicated. ( B ) RT-PCR analyses with primers specific to the mobE gene (left panel) or to the nrdA / mobE junction (right panel). <t>gDNA,</t> PCR with genomic <t>DNA;</t> RNA(-), RT-PCR reaction without added RNA; RT(-), reaction without added reverse transcriptase. ( C ) Northern blot analyses using probes specific to mobE (left panel) or to nrdA (right panel). Gene-specific probes were generated using primers ( 1 ) and ( 3 ) as indicated in panel (A) and listed in Table S1 .
    Phage Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore bacterial phage lambda dna
    mobE and nrdA are co-transcribed. ( A ) Schematic of the nrdA and mobE genes of phage T4, with promoters and putative RNA hairpin sequences indicated. The position of primers used for RT-PCR or northern blot analyses are indicated by arrows, and the length of the products indicated. ( B ) RT-PCR analyses with primers specific to the mobE gene (left panel) or to the nrdA / mobE junction (right panel). <t>gDNA,</t> PCR with genomic <t>DNA;</t> RNA(-), RT-PCR reaction without added RNA; RT(-), reaction without added reverse transcriptase. ( C ) Northern blot analyses using probes specific to mobE (left panel) or to nrdA (right panel). Gene-specific probes were generated using primers ( 1 ) and ( 3 ) as indicated in panel (A) and listed in Table S1 .
    Bacterial Phage Lambda Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore λ phage dna
    Fluorescence intensity and applied electrical potential vs time during electrokinetic injection of 5 μg∕mL YOYO-1 labeled <t>λ-phage</t> <t>DNA</t> in 40 mM TAE buffer across a single 300 nm diam×10 μm long PDMS nanopore.
    λ Phage Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Norgen Biotek phage dna
    Cleft-blocking domain occupies the <t>RNA-DNA</t> hybrid binding site in <t>phi14:2</t> RNAP gp66. a, b , and c , The structure of the active site of QDE-1 from N. crassa (PDB 2J7N 6 ), phi14:2 gp66, and T. thermophilus RNAP (PDB ID 2O5J 17 ), respectively. The active site of phi14:2 RNAP gp66 ( b ) is in a conformation incompatible with Mg binding.
    Phage Dna, supplied by Norgen Biotek, used in various techniques. Bioz Stars score: 99/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche bacteriophage λ dna
    In vitro assembly of empty and DNA-filled VLPs. ( a ) A scatter plot representing the height of empty ( black ) and DNA-filled ( blue ) VLPs at several time intervals is shown. Assembly of empty VLPs was induced by dialyzing VP1 pentamers against a high-ionic-strength buffer (2 M (NH 4 ) 2 SO 4 ), and for DNA-filled VLPs, pKYB1 (8.3 kbp) and <t>λ</t> DNA (48 kbp) were used to initiate assembly. All VLPs with a minimal diameter of 15 nm were included from a series of 1 μ m 2 images: 230 empty VLPs in 63 images and 490 DNA-filled VLPs in 86 images. ( b ) A histogram of the height of all VLPs from ( a ) is given, showing a more homogeneous population of ∼45 nm for DNA-filled VLPs and a more heterogeneous population ranging from 30 to 50 nm for empty VLPs. ( c – f ) Topographical three-dimensional images of assembled VLPs show structures with different heights after 4.5 h ( c ), which become larger, unorganized aggregates after 23 h ( d ). In the case of the DNA-filled VLPs, we observe several ∼45 nm VLPs after 3.5 h ( e ) and many ∼45 nm VLPs in a beads-on-a-string-like fashion after 23 h ( f ). To see this figure in color, go online.
    Bacteriophage λ Dna, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc phage dna
    Discovery of new Liddean-dependent interaction motifs for reptin. Next generation sequencing of peptide-phage pool obtained from a reptin screen in the apo and ligand bound state. Reptin was captured onto the solid phase without or with ligands ADP or Liddean. After selection of the peptide library on reptin protein, elution and propagation in bacteria, the phage <t>DNA</t> was amplified using <t>PCR</t> primer sets that capture the sequences flanking the peptide insert (as in Fig. S9 † ). Pooling of all phage into deep sequencing reactions can be done with subsequent deconvolution using the “bar code” whose position in the primer is indicated. (a) Parameters from the sequencing reactions from a representative screen are summarized. These include: (i) the sequencing reads before filtering non-specific binding peptides; (ii) the number of sequencing reads in apo or ligand bound protein; (iii) and the number of peptides that are shared in a number of apo or ligand bound screens. (b and c) Representative peptides that are enriched in the ligand bound state or suppressed in the ligand bound state are indicated to highlight a representative set of raw sequencing reads. (d) An example ciliopathy protein present in the list of human proteins which contain consensus sites identified by our Liddean-bound reptin screen. Processing the top 500 peptides from the apo and Liddean bound reptin using MEME to identify the top 10 consensus motifs (; http://meme.nbcr.net/meme/cgi-bin/meme.cgi) highlights the distinct sets of motifs acquired in the apo and ligand bound form. The motifs were processed using MAST or blastp to identify targets in the human proteome that have matches to these motifs, some of which are listed as potential ciliopathy targets.
    Phage Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 940 article reviews
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    92
    Thermo Fisher phage dna
    Discovery of new Liddean-dependent interaction motifs for reptin. Next generation sequencing of peptide-phage pool obtained from a reptin screen in the apo and ligand bound state. Reptin was captured onto the solid phase without or with ligands ADP or Liddean. After selection of the peptide library on reptin protein, elution and propagation in bacteria, the phage <t>DNA</t> was amplified using <t>PCR</t> primer sets that capture the sequences flanking the peptide insert (as in Fig. S9 † ). Pooling of all phage into deep sequencing reactions can be done with subsequent deconvolution using the “bar code” whose position in the primer is indicated. (a) Parameters from the sequencing reactions from a representative screen are summarized. These include: (i) the sequencing reads before filtering non-specific binding peptides; (ii) the number of sequencing reads in apo or ligand bound protein; (iii) and the number of peptides that are shared in a number of apo or ligand bound screens. (b and c) Representative peptides that are enriched in the ligand bound state or suppressed in the ligand bound state are indicated to highlight a representative set of raw sequencing reads. (d) An example ciliopathy protein present in the list of human proteins which contain consensus sites identified by our Liddean-bound reptin screen. Processing the top 500 peptides from the apo and Liddean bound reptin using MEME to identify the top 10 consensus motifs (; http://meme.nbcr.net/meme/cgi-bin/meme.cgi) highlights the distinct sets of motifs acquired in the apo and ligand bound form. The motifs were processed using MAST or blastp to identify targets in the human proteome that have matches to these motifs, some of which are listed as potential ciliopathy targets.
    Phage Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 543 article reviews
    Price from $9.99 to $1999.99
    phage dna - by Bioz Stars, 2020-08
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    Image Search Results


    Identification of a tranducing phage particle, φSaBov LUK , harboring linear phage DNA. (A) A schematic map of linear phage DNA, based on PCR results (see below). Coloring of genes is as in Fig. 1 . (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoR I restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1 ).

    Journal: Scientific Reports

    Article Title: Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island

    doi: 10.1038/srep09784

    Figure Lengend Snippet: Identification of a tranducing phage particle, φSaBov LUK , harboring linear phage DNA. (A) A schematic map of linear phage DNA, based on PCR results (see below). Coloring of genes is as in Fig. 1 . (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoR I restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1 ).

    Article Snippet: Phage DNA extraction and PCR The mitomycin C treated culture lysates were treated with excessive amounts of RNase and DNase I (Sigma-Aldrich, 100 unit each), and then phage particles were precipitated with NaCl (0.5 M final concentration) and polyethylene glycol 8000 (10%, wt/vol), followed by ultracentrifugation at 100,000 × g for 1 h. Phage DNA was extracted using DNeasy kit (Qiagen) according to the manufacturers' instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Southern Blot

    Heterogeneous excision products of the phage (φSaBov) that integrates at genomic island νSaβ. (A) A schematic map of νSaβ in the strain RF122. The arrows represent genes annotated in the GenBank entries 10 and colored based on key features. Orange; restriction modification system HsdR/M, yellow; serine protease cluster ( spl ), light green; bacteriocin gene cluster ( bsa ), pink; leukocidins ( lukD/E ), red; enterotoxin gene cluster ( egc ), cyan; genes related to phage. Direct repeat sequences associated with phage and those associated with the egc were annotated as attN L and attN R and attEGC L and attEGC R , respectively. Sequence variations in the direct repeats were underlined. Primers used for outward PCR and sequencing results of attN P and attEGC p were depicted. (B) Transmission electron microscope analysis of phage particles induced from the strain RF122. At least, three different head sizes (a, b, and c; 58, 47, 26 nm, respectively) of phages were observed. (C) Results of outward PCR using pInt/p1702 and p1693/p1759 for φSaBov N and φSaBov EGC , respectively. (D) RF122 chromosomal DNA (C) and phage DNA (P) were digested with EcoR I, separated by electrophoresis, and transferred to Nylon membrane for Southern blot analysis. Probes specific to the integrase gene (SAB1760, for φSaBov N ), SAB1737 (for φSaBov N and φSaBov EGC ), and the sem gene (for φSaBov EGC ) were used. (E) Phage spot test. Mitomycin C induced culture lysate from the strain RF122 (10 8 pfu/ml) was dropped onto the lawn culture of human ST36-SCCmecII (USA200), ST8-SCCmecIV (USA300), ST1-SCCmecIV (USA400), and bovine mastitis isolate (ST151).

    Journal: Scientific Reports

    Article Title: Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island

    doi: 10.1038/srep09784

    Figure Lengend Snippet: Heterogeneous excision products of the phage (φSaBov) that integrates at genomic island νSaβ. (A) A schematic map of νSaβ in the strain RF122. The arrows represent genes annotated in the GenBank entries 10 and colored based on key features. Orange; restriction modification system HsdR/M, yellow; serine protease cluster ( spl ), light green; bacteriocin gene cluster ( bsa ), pink; leukocidins ( lukD/E ), red; enterotoxin gene cluster ( egc ), cyan; genes related to phage. Direct repeat sequences associated with phage and those associated with the egc were annotated as attN L and attN R and attEGC L and attEGC R , respectively. Sequence variations in the direct repeats were underlined. Primers used for outward PCR and sequencing results of attN P and attEGC p were depicted. (B) Transmission electron microscope analysis of phage particles induced from the strain RF122. At least, three different head sizes (a, b, and c; 58, 47, 26 nm, respectively) of phages were observed. (C) Results of outward PCR using pInt/p1702 and p1693/p1759 for φSaBov N and φSaBov EGC , respectively. (D) RF122 chromosomal DNA (C) and phage DNA (P) were digested with EcoR I, separated by electrophoresis, and transferred to Nylon membrane for Southern blot analysis. Probes specific to the integrase gene (SAB1760, for φSaBov N ), SAB1737 (for φSaBov N and φSaBov EGC ), and the sem gene (for φSaBov EGC ) were used. (E) Phage spot test. Mitomycin C induced culture lysate from the strain RF122 (10 8 pfu/ml) was dropped onto the lawn culture of human ST36-SCCmecII (USA200), ST8-SCCmecIV (USA300), ST1-SCCmecIV (USA400), and bovine mastitis isolate (ST151).

    Article Snippet: Phage DNA extraction and PCR The mitomycin C treated culture lysates were treated with excessive amounts of RNase and DNase I (Sigma-Aldrich, 100 unit each), and then phage particles were precipitated with NaCl (0.5 M final concentration) and polyethylene glycol 8000 (10%, wt/vol), followed by ultracentrifugation at 100,000 × g for 1 h. Phage DNA was extracted using DNeasy kit (Qiagen) according to the manufacturers' instructions.

    Techniques: Modification, Sequencing, Polymerase Chain Reaction, Transmission Assay, Microscopy, Electrophoresis, Southern Blot, Spot Test

    Correlation between C q values of qPCR reaction and the amount of phage particles used as a template in qPCR reaction. Three independent serial dilutions ( N = 3) containing from 0 to 10 11 pfu/ml of 676Z phage, A3R phage, or T4 phage were used as templates in qPCR reactions. The X -axis presents the logarithm of phage titers used as template DNA in the qPCR reaction and the Y -axis presents C q values of the qPCR reactions.

    Journal: Frontiers in Microbiology

    Article Title: Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

    doi: 10.3389/fmicb.2017.02170

    Figure Lengend Snippet: Correlation between C q values of qPCR reaction and the amount of phage particles used as a template in qPCR reaction. Three independent serial dilutions ( N = 3) containing from 0 to 10 11 pfu/ml of 676Z phage, A3R phage, or T4 phage were used as templates in qPCR reactions. The X -axis presents the logarithm of phage titers used as template DNA in the qPCR reaction and the Y -axis presents C q values of the qPCR reactions.

    Article Snippet: Isolation of Genomic DNA from T4, A3R, and 676Z Phages and Preparation of DNA Standards Phage genomic DNA was isolated using GenElute Mammalian Genomic DNA Miniprep (Sigma–Aldrich, Poznan, Poland).

    Techniques: Real-time Polymerase Chain Reaction

    mobE and nrdA are co-transcribed. ( A ) Schematic of the nrdA and mobE genes of phage T4, with promoters and putative RNA hairpin sequences indicated. The position of primers used for RT-PCR or northern blot analyses are indicated by arrows, and the length of the products indicated. ( B ) RT-PCR analyses with primers specific to the mobE gene (left panel) or to the nrdA / mobE junction (right panel). gDNA, PCR with genomic DNA; RNA(-), RT-PCR reaction without added RNA; RT(-), reaction without added reverse transcriptase. ( C ) Northern blot analyses using probes specific to mobE (left panel) or to nrdA (right panel). Gene-specific probes were generated using primers ( 1 ) and ( 3 ) as indicated in panel (A) and listed in Table S1 .

    Journal: Nucleic Acids Research

    Article Title: Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII

    doi: 10.1093/nar/gkp769

    Figure Lengend Snippet: mobE and nrdA are co-transcribed. ( A ) Schematic of the nrdA and mobE genes of phage T4, with promoters and putative RNA hairpin sequences indicated. The position of primers used for RT-PCR or northern blot analyses are indicated by arrows, and the length of the products indicated. ( B ) RT-PCR analyses with primers specific to the mobE gene (left panel) or to the nrdA / mobE junction (right panel). gDNA, PCR with genomic DNA; RNA(-), RT-PCR reaction without added RNA; RT(-), reaction without added reverse transcriptase. ( C ) Northern blot analyses using probes specific to mobE (left panel) or to nrdA (right panel). Gene-specific probes were generated using primers ( 1 ) and ( 3 ) as indicated in panel (A) and listed in Table S1 .

    Article Snippet: Phage DNA was isolated using a Sigma gDNA kit (Sigma), following the gram-negative bacterial genomic DNA isolation protocol.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Polymerase Chain Reaction, Generated

    Mapping of MobE-generated nicks in vivo . ( A ) Schematic of the 5′ region of the nrdB gene showing the position of the primers used, the position of the DraI cleavage and MobE nicking sites, and the predicted lengths of the primer extension products. ( B ) Representative gel of primer extension reactions on the non-coding strand of DNA isolated at various time points (min) from co-infections with wild-type phages, or isolated from infections with T4 mobE deletion phage and T2. The sizes of a 100-bp ladder are labeled on the left edge of the gel, and the asterisk indicates the MobE nicking site present in wild-type but not mutant phage infections. −, no DNA added to primer extension; DraI, primer extension on DraI-digested gDNA. ( C ) Primer extension reactions on the coding strand, labeled as (B). For this representative gel, the DraI primer extension reaction was diluted 3-fold prior to electrophoresis. Note that the predicted size of the primer extension product is ∼78 nt.

    Journal: Nucleic Acids Research

    Article Title: Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII

    doi: 10.1093/nar/gkp769

    Figure Lengend Snippet: Mapping of MobE-generated nicks in vivo . ( A ) Schematic of the 5′ region of the nrdB gene showing the position of the primers used, the position of the DraI cleavage and MobE nicking sites, and the predicted lengths of the primer extension products. ( B ) Representative gel of primer extension reactions on the non-coding strand of DNA isolated at various time points (min) from co-infections with wild-type phages, or isolated from infections with T4 mobE deletion phage and T2. The sizes of a 100-bp ladder are labeled on the left edge of the gel, and the asterisk indicates the MobE nicking site present in wild-type but not mutant phage infections. −, no DNA added to primer extension; DraI, primer extension on DraI-digested gDNA. ( C ) Primer extension reactions on the coding strand, labeled as (B). For this representative gel, the DraI primer extension reaction was diluted 3-fold prior to electrophoresis. Note that the predicted size of the primer extension product is ∼78 nt.

    Article Snippet: Phage DNA was isolated using a Sigma gDNA kit (Sigma), following the gram-negative bacterial genomic DNA isolation protocol.

    Techniques: Generated, In Vivo, Isolation, Labeling, Mutagenesis, Electrophoresis

    Fluorescence intensity and applied electrical potential vs time during electrokinetic injection of 5 μg∕mL YOYO-1 labeled λ-phage DNA in 40 mM TAE buffer across a single 300 nm diam×10 μm long PDMS nanopore.

    Journal: Biomicrofluidics

    Article Title: Single nanopore transport of synthetic and biological polyelectrolytes in three-dimensional hybrid microfluidic/nanofluidic devices

    doi: 10.1063/1.3059546

    Figure Lengend Snippet: Fluorescence intensity and applied electrical potential vs time during electrokinetic injection of 5 μg∕mL YOYO-1 labeled λ-phage DNA in 40 mM TAE buffer across a single 300 nm diam×10 μm long PDMS nanopore.

    Article Snippet: Polystyrene sulfonate sodium salt (PSS), polyallylamine chloride salt (PAA), tris(hydroxymethyl)aminomethane (TRIS), ethylenediaminetetraacetic (EDTA), λ-phage DNA, and acetic acid (Sigma Aldrich Co., St. Louis, MO) and YOYO-1 (Invitrogen, Carlsbad, CA) were used as received.

    Techniques: Fluorescence, Injection, Labeling

    Cleft-blocking domain occupies the RNA-DNA hybrid binding site in phi14:2 RNAP gp66. a, b , and c , The structure of the active site of QDE-1 from N. crassa (PDB 2J7N 6 ), phi14:2 gp66, and T. thermophilus RNAP (PDB ID 2O5J 17 ), respectively. The active site of phi14:2 RNAP gp66 ( b ) is in a conformation incompatible with Mg binding.

    Journal: bioRxiv

    Article Title: Structure and function of virion RNA polymerase of crAss-like phage

    doi: 10.1101/2020.03.07.982082

    Figure Lengend Snippet: Cleft-blocking domain occupies the RNA-DNA hybrid binding site in phi14:2 RNAP gp66. a, b , and c , The structure of the active site of QDE-1 from N. crassa (PDB 2J7N 6 ), phi14:2 gp66, and T. thermophilus RNAP (PDB ID 2O5J 17 ), respectively. The active site of phi14:2 RNAP gp66 ( b ) is in a conformation incompatible with Mg binding.

    Article Snippet: DNA templates for transcription assay For phi14:2 RNAP transcription assay genomic DNA of phi14:2 was purified using the Phage DNA Isolation Kit (Norgen Biotek Corp) according to the manufacturer’s instructions.

    Techniques: Blocking Assay, Binding Assay

    Global analysis of phi14:2 transcription during phi14:2 infection. a , Schematics of the phi14:2 genome 3 . ORFs are marked as arrows and numbered according to the study by Yutin et al 2 (Supplementary Table 1). Intergenic regions larger than 50 base pairs are shown as grey rectangles. Replicative, gene expression, and capsid gene modules are marked by green, violet, and blue dashed frames, correspondingly 2 . Early, middle, and late genes are colored green, purple, and blue, correspondingly. Heat maps indicating the temporal pattern of phi14:2 transcripts and relative abundance of phi14:2 transcripts are shown below the genome. In the top heat map, the transcript abundance for each gene or intergenic region longer than 50 base pairs is normalized to the maximum transcript abundance for this particular gene/intergenic region; In the bottom heat map, the same quantities were normalized to the absolute maximum that corresponded to the abundance of the late gene g091 (major capsid protein) at 190 min post infection. b , Time courses of accumulation of individual phi14:2 transcripts divided into three temporal classes during infection; the y axis shows abundance of individual genes transcripts normalized to the maximal value for this gene obtained in Rif-libraries. c , Transcription by gp66 of denatured phi14:2 DNA and by C. baltica RNAP of a PCR-fragment containing the T7 A1 promoter in the absence and in the presence of rifampicin. d , Time courses of accumulation of early, middle, and late phi14:2 transcripts during infection in the presence of rifampicin; the y axis shows abundance of individual genes transcripts in Rif+ libraries normalized to maximal value for this gene obtained in Rif-libraries. e , WebLogos of phi14:2 middle promoters located upstream of middle genes g070, g075, g108, and g110 (top panel) and cumulative consensus of 126 crAss-like phage middle/late promoters (bottom panel, Supplementary file 1).

    Journal: bioRxiv

    Article Title: Structure and function of virion RNA polymerase of crAss-like phage

    doi: 10.1101/2020.03.07.982082

    Figure Lengend Snippet: Global analysis of phi14:2 transcription during phi14:2 infection. a , Schematics of the phi14:2 genome 3 . ORFs are marked as arrows and numbered according to the study by Yutin et al 2 (Supplementary Table 1). Intergenic regions larger than 50 base pairs are shown as grey rectangles. Replicative, gene expression, and capsid gene modules are marked by green, violet, and blue dashed frames, correspondingly 2 . Early, middle, and late genes are colored green, purple, and blue, correspondingly. Heat maps indicating the temporal pattern of phi14:2 transcripts and relative abundance of phi14:2 transcripts are shown below the genome. In the top heat map, the transcript abundance for each gene or intergenic region longer than 50 base pairs is normalized to the maximum transcript abundance for this particular gene/intergenic region; In the bottom heat map, the same quantities were normalized to the absolute maximum that corresponded to the abundance of the late gene g091 (major capsid protein) at 190 min post infection. b , Time courses of accumulation of individual phi14:2 transcripts divided into three temporal classes during infection; the y axis shows abundance of individual genes transcripts normalized to the maximal value for this gene obtained in Rif-libraries. c , Transcription by gp66 of denatured phi14:2 DNA and by C. baltica RNAP of a PCR-fragment containing the T7 A1 promoter in the absence and in the presence of rifampicin. d , Time courses of accumulation of early, middle, and late phi14:2 transcripts during infection in the presence of rifampicin; the y axis shows abundance of individual genes transcripts in Rif+ libraries normalized to maximal value for this gene obtained in Rif-libraries. e , WebLogos of phi14:2 middle promoters located upstream of middle genes g070, g075, g108, and g110 (top panel) and cumulative consensus of 126 crAss-like phage middle/late promoters (bottom panel, Supplementary file 1).

    Article Snippet: DNA templates for transcription assay For phi14:2 RNAP transcription assay genomic DNA of phi14:2 was purified using the Phage DNA Isolation Kit (Norgen Biotek Corp) according to the manufacturer’s instructions.

    Techniques: Infection, Expressing, Polymerase Chain Reaction

    In vitro transcription activity of the phi14:2 RNAP gp66. a , Transcription by gp66 of genomic DNA of phages phi14:2 and M13 (double- and single-stranded forms) at 30, 22, and 10°C; the reaction products were resolved by electrophoresis in 5 % (w/v) denaturing 8 M urea polyacrylamide gel and revealed by autoradiography. b , Transcription by gp66 of native and denatured genomic DNA of phage phi14:2. c , Completed transcription reactions of phi14:2 genomic DNA were treated with DNase RQ1 or RNase T1 prior to loading on the gel. d , Activity of gp66 requires Mg ions and ATP. Denatured genomic DNA of phi14:2 phage has been used as a template. e , Transcription of phi14:2 denatured genomic DNA by wild-type gp66 and gp66 mutants carrying single alanine substitutions of each aspartate in the DFDID motif.

    Journal: bioRxiv

    Article Title: Structure and function of virion RNA polymerase of crAss-like phage

    doi: 10.1101/2020.03.07.982082

    Figure Lengend Snippet: In vitro transcription activity of the phi14:2 RNAP gp66. a , Transcription by gp66 of genomic DNA of phages phi14:2 and M13 (double- and single-stranded forms) at 30, 22, and 10°C; the reaction products were resolved by electrophoresis in 5 % (w/v) denaturing 8 M urea polyacrylamide gel and revealed by autoradiography. b , Transcription by gp66 of native and denatured genomic DNA of phage phi14:2. c , Completed transcription reactions of phi14:2 genomic DNA were treated with DNase RQ1 or RNase T1 prior to loading on the gel. d , Activity of gp66 requires Mg ions and ATP. Denatured genomic DNA of phi14:2 phage has been used as a template. e , Transcription of phi14:2 denatured genomic DNA by wild-type gp66 and gp66 mutants carrying single alanine substitutions of each aspartate in the DFDID motif.

    Article Snippet: DNA templates for transcription assay For phi14:2 RNAP transcription assay genomic DNA of phi14:2 was purified using the Phage DNA Isolation Kit (Norgen Biotek Corp) according to the manufacturer’s instructions.

    Techniques: In Vitro, Activity Assay, Electrophoresis, Autoradiography

    In vitro assembly of empty and DNA-filled VLPs. ( a ) A scatter plot representing the height of empty ( black ) and DNA-filled ( blue ) VLPs at several time intervals is shown. Assembly of empty VLPs was induced by dialyzing VP1 pentamers against a high-ionic-strength buffer (2 M (NH 4 ) 2 SO 4 ), and for DNA-filled VLPs, pKYB1 (8.3 kbp) and λ DNA (48 kbp) were used to initiate assembly. All VLPs with a minimal diameter of 15 nm were included from a series of 1 μ m 2 images: 230 empty VLPs in 63 images and 490 DNA-filled VLPs in 86 images. ( b ) A histogram of the height of all VLPs from ( a ) is given, showing a more homogeneous population of ∼45 nm for DNA-filled VLPs and a more heterogeneous population ranging from 30 to 50 nm for empty VLPs. ( c – f ) Topographical three-dimensional images of assembled VLPs show structures with different heights after 4.5 h ( c ), which become larger, unorganized aggregates after 23 h ( d ). In the case of the DNA-filled VLPs, we observe several ∼45 nm VLPs after 3.5 h ( e ) and many ∼45 nm VLPs in a beads-on-a-string-like fashion after 23 h ( f ). To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Effect of dsDNA on the Assembly Pathway and Mechanical Strength of SV40 VP1 Virus-like Particles

    doi: 10.1016/j.bpj.2018.07.044

    Figure Lengend Snippet: In vitro assembly of empty and DNA-filled VLPs. ( a ) A scatter plot representing the height of empty ( black ) and DNA-filled ( blue ) VLPs at several time intervals is shown. Assembly of empty VLPs was induced by dialyzing VP1 pentamers against a high-ionic-strength buffer (2 M (NH 4 ) 2 SO 4 ), and for DNA-filled VLPs, pKYB1 (8.3 kbp) and λ DNA (48 kbp) were used to initiate assembly. All VLPs with a minimal diameter of 15 nm were included from a series of 1 μ m 2 images: 230 empty VLPs in 63 images and 490 DNA-filled VLPs in 86 images. ( b ) A histogram of the height of all VLPs from ( a ) is given, showing a more homogeneous population of ∼45 nm for DNA-filled VLPs and a more heterogeneous population ranging from 30 to 50 nm for empty VLPs. ( c – f ) Topographical three-dimensional images of assembled VLPs show structures with different heights after 4.5 h ( c ), which become larger, unorganized aggregates after 23 h ( d ). In the case of the DNA-filled VLPs, we observe several ∼45 nm VLPs after 3.5 h ( e ) and many ∼45 nm VLPs in a beads-on-a-string-like fashion after 23 h ( f ). To see this figure in color, go online.

    Article Snippet: Bacteriophage λ DNA of 48,502 bp (Roche Diagnostics, Mannheim, Germany) was used at concentrations of 5–158 ng/ μ L DNA per 360–440 nM VP1 pentamers (72:1, 360:1, and 2750:1 ratios) and incubated at room temperature for 2, 3, 6, or 24 h or 90 min on ice followed by 2 or 24 h at room temperature.

    Techniques: In Vitro

    Determination of genome ends of UAB_Phi20 phage after digestion with EcoRI enzyme . Genome of bacteriophage P22 digested with Eco RI was used as control. Arrows indicate the 4007-bp fragment containing the pac sequence. Lambda DNA digested with Hin dIII (M1) or Bst EII (M2) were used as molecular markers. Sizes (bp) are indicated on both sides of the image.

    Journal: Frontiers in Microbiology

    Article Title: Genomics of Three New Bacteriophages Useful in the Biocontrol of Salmonella

    doi: 10.3389/fmicb.2016.00545

    Figure Lengend Snippet: Determination of genome ends of UAB_Phi20 phage after digestion with EcoRI enzyme . Genome of bacteriophage P22 digested with Eco RI was used as control. Arrows indicate the 4007-bp fragment containing the pac sequence. Lambda DNA digested with Hin dIII (M1) or Bst EII (M2) were used as molecular markers. Sizes (bp) are indicated on both sides of the image.

    Article Snippet: Lambda bacteriophage DNA (Roche Diagnostics GmbH, Germany) with cohesive ends and treated with the same methodology served as a control.

    Techniques: Sequencing, Lambda DNA Preparation

    Discovery of new Liddean-dependent interaction motifs for reptin. Next generation sequencing of peptide-phage pool obtained from a reptin screen in the apo and ligand bound state. Reptin was captured onto the solid phase without or with ligands ADP or Liddean. After selection of the peptide library on reptin protein, elution and propagation in bacteria, the phage DNA was amplified using PCR primer sets that capture the sequences flanking the peptide insert (as in Fig. S9 † ). Pooling of all phage into deep sequencing reactions can be done with subsequent deconvolution using the “bar code” whose position in the primer is indicated. (a) Parameters from the sequencing reactions from a representative screen are summarized. These include: (i) the sequencing reads before filtering non-specific binding peptides; (ii) the number of sequencing reads in apo or ligand bound protein; (iii) and the number of peptides that are shared in a number of apo or ligand bound screens. (b and c) Representative peptides that are enriched in the ligand bound state or suppressed in the ligand bound state are indicated to highlight a representative set of raw sequencing reads. (d) An example ciliopathy protein present in the list of human proteins which contain consensus sites identified by our Liddean-bound reptin screen. Processing the top 500 peptides from the apo and Liddean bound reptin using MEME to identify the top 10 consensus motifs (; http://meme.nbcr.net/meme/cgi-bin/meme.cgi) highlights the distinct sets of motifs acquired in the apo and ligand bound form. The motifs were processed using MAST or blastp to identify targets in the human proteome that have matches to these motifs, some of which are listed as potential ciliopathy targets.

    Journal: Chemical Science

    Article Title: Discovery of a novel ligand that modulates the protein–protein interactions of the AAA+ superfamily oncoprotein reptin of a novel ligand that modulates the protein–protein interactions of the AAA+ superfamily oncoprotein reptin †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc03885aClick here for additional data file.

    doi: 10.1039/c4sc03885a

    Figure Lengend Snippet: Discovery of new Liddean-dependent interaction motifs for reptin. Next generation sequencing of peptide-phage pool obtained from a reptin screen in the apo and ligand bound state. Reptin was captured onto the solid phase without or with ligands ADP or Liddean. After selection of the peptide library on reptin protein, elution and propagation in bacteria, the phage DNA was amplified using PCR primer sets that capture the sequences flanking the peptide insert (as in Fig. S9 † ). Pooling of all phage into deep sequencing reactions can be done with subsequent deconvolution using the “bar code” whose position in the primer is indicated. (a) Parameters from the sequencing reactions from a representative screen are summarized. These include: (i) the sequencing reads before filtering non-specific binding peptides; (ii) the number of sequencing reads in apo or ligand bound protein; (iii) and the number of peptides that are shared in a number of apo or ligand bound screens. (b and c) Representative peptides that are enriched in the ligand bound state or suppressed in the ligand bound state are indicated to highlight a representative set of raw sequencing reads. (d) An example ciliopathy protein present in the list of human proteins which contain consensus sites identified by our Liddean-bound reptin screen. Processing the top 500 peptides from the apo and Liddean bound reptin using MEME to identify the top 10 consensus motifs (; http://meme.nbcr.net/meme/cgi-bin/meme.cgi) highlights the distinct sets of motifs acquired in the apo and ligand bound form. The motifs were processed using MAST or blastp to identify targets in the human proteome that have matches to these motifs, some of which are listed as potential ciliopathy targets.

    Article Snippet: Polymerase chain reaction (PCR) and deep sequencing of phage was carried out in the following stages; (i) PCR was used to amplify phage DNA from each round of screening using the primer bar codes in table code (Table S3 ) that have an Illumina adaptor sequence and a 3 letter bar code; (ii) equal amounts of DNA was gel purified on a 2% agarose gel to create a pool (5 µg) that was sequenced by Otogenetics (USA).

    Techniques: Next-Generation Sequencing, Selection, Amplification, Polymerase Chain Reaction, Sequencing, Binding Assay