xbai  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    New England Biolabs xbai
    Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai/product/New England Biolabs
    Average 99 stars, based on 312 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2020-04
    99/100 stars

    Images

    Related Articles

    Clone Assay:

    Article Title: Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling
    Article Snippet: .. The PCR product was digested with EcoRI and XbaI (NEW ENGLAND Biolabs Inc., catalog #R0101L and #R0145L), purified with a MinElute PCR Purification Kit (QIAGEN, catalog #28006), and subsequently cloned into an EcoRI- and XbaI -linearized pCS2+ vector using the T & A cloning kit (Yeastern Biotech, catalog # YC001) and TaKaRa DNA ligation kit (TAKARA BIO INC, catalog #6024). ..

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: .. Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice. .. Plasmid construction The gene for the Gag/p24 capsid protein of HIV-1 was amplified from a plasmid containing the proviral genome of the HIV strain HxB2 by PCR using primers 59-TAAGAATTCCCTATAGTGCAG AACATCCAGG-39 (forward, EcoRI site in italics) and 59-GCTTCTAG ACAAAACTCTTGCCTTATGGC-39 (reverse, XbaI site in italics).

    Amplification:

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice. .. Plasmid construction The gene for the Gag/p24 capsid protein of HIV-1 was amplified from a plasmid containing the proviral genome of the HIV strain HxB2 by PCR using primers 59-TAAGAATTCCCTATAGTGCAG AACATCCAGG-39 (forward, EcoRI site in italics) and 59-GCTTCTAG ACAAAACTCTTGCCTTATGGC-39 (reverse, XbaI site in italics).

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.
    Article Snippet: Confirmation of RSV subgroup A and B RT-LAMP products were purified using the QIAquick PCR Purification Kit (Qiagen), and the purified amplicons were digested with the restriction enzyme, NlaIII or XbaI, under optimal conditions (New England Biolabs, Ipswich, MA, USA). .. The amplicon from subgroup A RSV was digested with NlaIII, while that from subgroup B RSV was digested with XbaI.

    Agarose Gel Electrophoresis:

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.
    Article Snippet: Confirmation of RSV subgroup A and B RT-LAMP products were purified using the QIAquick PCR Purification Kit (Qiagen), and the purified amplicons were digested with the restriction enzyme, NlaIII or XbaI, under optimal conditions (New England Biolabs, Ipswich, MA, USA). .. After 2 h of digestion, RT-LAMP products were separated by electrophoresis on a 3% agarose gel, stained with ethidium bromide, and the bands were visualized under ultraviolet light.

    DNA Ligation:

    Article Title: Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling
    Article Snippet: .. The PCR product was digested with EcoRI and XbaI (NEW ENGLAND Biolabs Inc., catalog #R0101L and #R0145L), purified with a MinElute PCR Purification Kit (QIAGEN, catalog #28006), and subsequently cloned into an EcoRI- and XbaI -linearized pCS2+ vector using the T & A cloning kit (Yeastern Biotech, catalog # YC001) and TaKaRa DNA ligation kit (TAKARA BIO INC, catalog #6024). ..

    Ligation:

    Article Title: Ca2+ transients in melanocyte dendrites and dendritic spine-like structures evoked by cell-to-cell signaling.
    Article Snippet: .. Melanocytes are the neural crest-derived pigment-producing cells of the skin that possess dendrites. .. Melanocytes are the neural crest-derived pigment-producing cells of the skin that possess dendrites.

    Construct:

    Article Title: Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling
    Article Snippet: Paragraph title: DNA constructs: cloning and vectors ... The PCR product was digested with EcoRI and XbaI (NEW ENGLAND Biolabs Inc., catalog #R0101L and #R0145L), purified with a MinElute PCR Purification Kit (QIAGEN, catalog #28006), and subsequently cloned into an EcoRI- and XbaI -linearized pCS2+ vector using the T & A cloning kit (Yeastern Biotech, catalog # YC001) and TaKaRa DNA ligation kit (TAKARA BIO INC, catalog #6024).

    Purification:

    Article Title: Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling
    Article Snippet: .. The PCR product was digested with EcoRI and XbaI (NEW ENGLAND Biolabs Inc., catalog #R0101L and #R0145L), purified with a MinElute PCR Purification Kit (QIAGEN, catalog #28006), and subsequently cloned into an EcoRI- and XbaI -linearized pCS2+ vector using the T & A cloning kit (Yeastern Biotech, catalog # YC001) and TaKaRa DNA ligation kit (TAKARA BIO INC, catalog #6024). ..

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.
    Article Snippet: .. Confirmation of RSV subgroup A and B RT-LAMP products were purified using the QIAquick PCR Purification Kit (Qiagen), and the purified amplicons were digested with the restriction enzyme, NlaIII or XbaI, under optimal conditions (New England Biolabs, Ipswich, MA, USA). .. After 2 h of digestion, RT-LAMP products were separated by electrophoresis on a 3% agarose gel, stained with ethidium bromide, and the bands were visualized under ultraviolet light.

    Electrophoresis:

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.
    Article Snippet: Confirmation of RSV subgroup A and B RT-LAMP products were purified using the QIAquick PCR Purification Kit (Qiagen), and the purified amplicons were digested with the restriction enzyme, NlaIII or XbaI, under optimal conditions (New England Biolabs, Ipswich, MA, USA). .. After 2 h of digestion, RT-LAMP products were separated by electrophoresis on a 3% agarose gel, stained with ethidium bromide, and the bands were visualized under ultraviolet light.

    Modification:

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice. .. The amplification products were cloned in the EcoRI and XbaI sites of the modified pMALc2X vector resulting in open reading frames for fusion proteins containing the E. coli maltose binding protein (MBP), Gag/p24 and the C-terminal SARS-N peptide (Fig. 1 ).

    Polymerase Chain Reaction:

    Article Title: Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling
    Article Snippet: .. The PCR product was digested with EcoRI and XbaI (NEW ENGLAND Biolabs Inc., catalog #R0101L and #R0145L), purified with a MinElute PCR Purification Kit (QIAGEN, catalog #28006), and subsequently cloned into an EcoRI- and XbaI -linearized pCS2+ vector using the T & A cloning kit (Yeastern Biotech, catalog # YC001) and TaKaRa DNA ligation kit (TAKARA BIO INC, catalog #6024). ..

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice. .. Plasmid construction The gene for the Gag/p24 capsid protein of HIV-1 was amplified from a plasmid containing the proviral genome of the HIV strain HxB2 by PCR using primers 59-TAAGAATTCCCTATAGTGCAG AACATCCAGG-39 (forward, EcoRI site in italics) and 59-GCTTCTAG ACAAAACTCTTGCCTTATGGC-39 (reverse, XbaI site in italics).

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.
    Article Snippet: .. Confirmation of RSV subgroup A and B RT-LAMP products were purified using the QIAquick PCR Purification Kit (Qiagen), and the purified amplicons were digested with the restriction enzyme, NlaIII or XbaI, under optimal conditions (New England Biolabs, Ipswich, MA, USA). .. After 2 h of digestion, RT-LAMP products were separated by electrophoresis on a 3% agarose gel, stained with ethidium bromide, and the bands were visualized under ultraviolet light.

    Expressing:

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: To generate expression plasmids, we used oligonucleotides for the coding and non-coding strand of the SARS-N peptides with the following sequences: epitope of 8G7: 59-CTAGAGGTAATTCAAGAAATTCAACTCCTGGCAGCTAGCTGCA-39 (sense) and 59-GCTAGCTGCCAGGAGTTGAATTTCTTGAATTACCT-39 (antisense) (sequences in italics mark the XbaI and PstI overhang, stop codon is indicated in bold); epitope of 2G8: 59-CTAGAAAAAAG AAAAAGACTGATGAAGCTCAGCCTTAGCTGCA-39 and 59-GCTAAGGCT GAGCTTCATCAGTCTTTTTCTTTTTT-39. .. Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice.

    Sequencing:

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: Plasmid construction The mAb AMII/8G7B2C (8G7) recognizes the amino acid sequence GNSRNSTPGS (amino acid positions 193 to 202). .. Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice.

    Staining:

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification.
    Article Snippet: Confirmation of RSV subgroup A and B RT-LAMP products were purified using the QIAquick PCR Purification Kit (Qiagen), and the purified amplicons were digested with the restriction enzyme, NlaIII or XbaI, under optimal conditions (New England Biolabs, Ipswich, MA, USA). .. After 2 h of digestion, RT-LAMP products were separated by electrophoresis on a 3% agarose gel, stained with ethidium bromide, and the bands were visualized under ultraviolet light.

    Binding Assay:

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice. .. The amplification products were cloned in the EcoRI and XbaI sites of the modified pMALc2X vector resulting in open reading frames for fusion proteins containing the E. coli maltose binding protein (MBP), Gag/p24 and the C-terminal SARS-N peptide (Fig. 1 ).

    Plasmid Preparation:

    Article Title: Zebrafish WNK Lysine Deficient Protein Kinase 1 (wnk1) Affects Angiogenesis Associated with VEGF Signaling
    Article Snippet: .. The PCR product was digested with EcoRI and XbaI (NEW ENGLAND Biolabs Inc., catalog #R0101L and #R0145L), purified with a MinElute PCR Purification Kit (QIAGEN, catalog #28006), and subsequently cloned into an EcoRI- and XbaI -linearized pCS2+ vector using the T & A cloning kit (Yeastern Biotech, catalog # YC001) and TaKaRa DNA ligation kit (TAKARA BIO INC, catalog #6024). ..

    Article Title: Peptides from the SARS-associated coronavirus as tags for protein expression and purification.
    Article Snippet: .. Oligonucleotides were hybridized and cloned in the XbaI and PstI sites of vector pMAL TM c2X (New England Biolabs) for C-terminal fusion with the protein of choice. .. Plasmid construction The gene for the Gag/p24 capsid protein of HIV-1 was amplified from a plasmid containing the proviral genome of the HIV strain HxB2 by PCR using primers 59-TAAGAATTCCCTATAGTGCAG AACATCCAGG-39 (forward, EcoRI site in italics) and 59-GCTTCTAG ACAAAACTCTTGCCTTATGGC-39 (reverse, XbaI site in italics).

    Article Title: Ca2+ transients in melanocyte dendrites and dendritic spine-like structures evoked by cell-to-cell signaling.
    Article Snippet: .. Melanocytes are the neural crest-derived pigment-producing cells of the skin that possess dendrites. .. Melanocytes are the neural crest-derived pigment-producing cells of the skin that possess dendrites.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs restriction endonucleases xbai
    (A) <t>PFGE</t> analysis of S . Typhimurium DT104 using <t>XbaI</t> and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,
    Restriction Endonucleases Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonucleases xbai/product/New England Biolabs
    Average 99 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    restriction endonucleases xbai - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    New England Biolabs xbai site located
    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a <t>XbaI</t> recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the <t>‘GAA</t> orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.
    Xbai Site Located, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai site located/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xbai site located - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    86
    New England Biolabs algr d54e xbai r
    Phosphorylated <t>AlgR</t> and AlgR <t>D54E</t> bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.
    Algr D54e Xbai R, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/algr d54e xbai r/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    algr d54e xbai r - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    Image Search Results


    (A) PFGE analysis of S . Typhimurium DT104 using XbaI and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,

    Journal: Applied and Environmental Microbiology

    Article Title: Evidence of Metabolic Switching and Implications for Food Safety from the Phenome(s) of Salmonella enterica Serovar Typhimurium DT104 Cultured at Selected Points across the Pork Production Food Chain

    doi: 10.1128/AEM.01041-13

    Figure Lengend Snippet: (A) PFGE analysis of S . Typhimurium DT104 using XbaI and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,

    Article Snippet: Pulsed-field gel electrophoresis (PFGE) was performed using the restriction endonucleases XbaI and AvrII (New England BioLabs) by following the CDC PulseNet protocol ( ).

    Techniques:

    Southern blot showing copy numbers of the 16D10i-2 RNAi transgene in transformed potato lines and controls. For each plant line, 15 µg DNA was digested with 50 U Xba I and separated on a 0.8% agarose gel. 82-4, PA99N82-4 advanced breeding line;

    Journal: Journal of Nematology

    Article Title: Broad Meloidogyne Resistance in Potato Based on RNA Interference of Effector Gene 16D10

    doi:

    Figure Lengend Snippet: Southern blot showing copy numbers of the 16D10i-2 RNAi transgene in transformed potato lines and controls. For each plant line, 15 µg DNA was digested with 50 U Xba I and separated on a 0.8% agarose gel. 82-4, PA99N82-4 advanced breeding line;

    Article Snippet: For each plant line, 15 µg DNA was digested with 50 U Xba I (New England Biolabs, Ipswich, MA) for 16 hr at 37 ° C, then separated on a 0.8% agarose gel at 70 V for 16 hr and transferred to a GeneScreen Plus nylon membrane (PerkinElmer, Boston, MA) in 10× saline sodium citrate (SSC) buffer (1.5 M NaCl, 0.15 M sodium citrate, pH 7).

    Techniques: Southern Blot, Transformation Assay, Agarose Gel Electrophoresis

    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Construct, Sequencing, Clone Assay

    DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Transformation Assay, Mutagenesis, Construct, Derivative Assay

    DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Transformation Assay

    DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Polymerase Chain Reaction, Marker, Produced, Derivative Assay

    DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Construct, Produced, Transformation Assay, Mutagenesis, Derivative Assay, Plasmid Preparation

    Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: In Vitro, Binding Assay, Purification, Incubation

    AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Thin Layer Chromatography, Colorimetric Assay

    Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Negative Control

    AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Activity Assay