ptxb1  (New England Biolabs)


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    Name:
    pTXB1 Vector DNA
    Description:
    pTXB1 Vector DNA 10 ug
    Catalog Number:
    n6707s
    Price:
    133
    Size:
    10 ug
    Category:
    E coli Expression Vectors
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    Structured Review

    New England Biolabs ptxb1
    pTXB1 Vector DNA
    pTXB1 Vector DNA 10 ug
    https://www.bioz.com/result/ptxb1/product/New England Biolabs
    Average 93 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    ptxb1 - by Bioz Stars, 2020-02
    93/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    Article Title: BDNF pro-peptide regulates dendritic spines via caspase-3
    Article Snippet: .. DNA construct and purification of BDNF pro-peptide DNA sequence encoding the human BDNF pro-peptide (Val66 ) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between Nde I and Spe I sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. .. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S).

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: Paragraph title: Cloning of RTD-C3 and RTD-C7 and in vitro production of RTD-1 ... Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. DNA encoding Aβ1-29 was cloned into pTXB1 vector [ ], which utilizes an intein tag system. .. This vector was chosen based on its ability to generate a C-terminal thioester suitable for native chemical ligation after cleaving the intein tag using MESNA.

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: .. Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After selection on solid LB media containing ampicillin (100 µg/mL), individual colonies were used to inoculate 1 L of LB media containing ampicillin (100 µg/mL) and grown at 37°C to late log phase.

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: .. The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion. .. The CzrA 1–95-intein fusion was expressed in E. coli BL21(DE3) and after sonication in Buffer C (25 mM Tris, 0.5 M NaCl, 2 mM TCEP, pH 8.0), was found to remain in the low speed lysis pellet.

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: Paragraph title: Cloning and mutagenesis ... Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707).

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: Paragraph title: Cloning and in vitro expression of backbone cyclized Crp4 variants ... Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth.

    Article Title: Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System *
    Article Snippet: .. The DNA encoding 1–76 amino acids of Rub1 was cloned into pTXB1 vector (NEB) using standard techniques, and the construct was verified by DNA sequencing. .. Details of the cloning procedure and primer sequences are provided in the supplemental information.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Centrifugation:

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After incubating overnight at 16°C, cells were harvested by centrifugation, resuspended in 25 mL of 10 mM Tris-HCl, 500 mM NaCl buffer, pH 8.0 (Buffer A) and sonicated at 4°C.

    Amplification:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: The amplified DNA fragment was purified on agarose gel and ligated into pGEM-T vector (Promega) using A–T base pairing. .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD).

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). ..

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Synthesized:

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: The C-terminal peptide of CzrA (residues 96–106, H96 substituted with Cys) was synthesized with incorporation of MeH (1-methylhistidine) obtained as a Boc-derivative (Bachem, CA) at residue 97 using Boc-based solid phase peptide synthesis. .. The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion.

    Construct:

    Article Title: BDNF pro-peptide regulates dendritic spines via caspase-3
    Article Snippet: .. DNA construct and purification of BDNF pro-peptide DNA sequence encoding the human BDNF pro-peptide (Val66 ) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between Nde I and Spe I sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. .. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S).

    Article Title: Brain-derived neurotrophic factor propeptide inhibits proliferation and induces apoptosis in C6 glioma cells
    Article Snippet: .. DNA construct and purification of the BDNF propeptide A DNA sequence that encodes for the human BDNF propeptide up to the furin cleavage site was inserted between the NdeI and SpeI sites of a pTXB1 vector (New England Biolab, Ipswich, Massachusetts, USA). .. Recombinant BDNF propeptide was expressed in BL21 Escherichia coli and purified using an IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) kit according to the manufacturer’s protocol (cat. #E6901S; New England Biolab).

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). ..

    Article Title: Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System *
    Article Snippet: .. The DNA encoding 1–76 amino acids of Rub1 was cloned into pTXB1 vector (NEB) using standard techniques, and the construct was verified by DNA sequencing. .. Details of the cloning procedure and primer sequences are provided in the supplemental information.

    Incubation:

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. The soluble fraction was incubated with chitin beads (New England Biolabs) for 1 h at 4°C and the beads were washed with column buffer (0.1 mM EDTA, 50 mM sodium phosphate, 250 mM sodium chloride buffer at pH 7.2) containing 0.1% Triton X-100 followed by washes with column buffer without Triton X-100.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. The mixture was heated to 42 °C for 30 s after which 475 μL LB medium was added followed by incubation at 37 °C for 1 h. 100 μL was spread on LB-Ampicilin+ agar plates and incubated overnight at 37 °C.

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. The soluble fraction was incubated with chitin beads (New England Biolabs) for 1 h at 4°C and the beads were washed with column buffer (0.1 mM EDTA, 50 mM sodium phosphate, 250 mM sodium chloride buffer at pH 7.2) containing 0.1% Triton X-100 followed by washes with column buffer without Triton X-100.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. The mixture was heated to 42 °C for 30 s after which 475 μL LB medium was added followed by incubation at 37 °C for 1 h. 100 μL was spread on LB-Ampicilin+ agar plates and incubated overnight at 37 °C.

    Activity Assay:

    Article Title: Brain-derived neurotrophic factor propeptide inhibits proliferation and induces apoptosis in C6 glioma cells
    Article Snippet: DNA construct and purification of the BDNF propeptide A DNA sequence that encodes for the human BDNF propeptide up to the furin cleavage site was inserted between the NdeI and SpeI sites of a pTXB1 vector (New England Biolab, Ipswich, Massachusetts, USA). .. The IMPACT system utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag.

    Expressing:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: Paragraph title: 2.1. Cloning, expression and purification ... After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD).

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: .. 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. This plasmid was transformed into T7 Express E. coli cells (New England Biolabs).

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: .. Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After selection on solid LB media containing ampicillin (100 µg/mL), individual colonies were used to inoculate 1 L of LB media containing ampicillin (100 µg/mL) and grown at 37°C to late log phase.

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: Cloning and mutagenesis Cloning and expression of 6xHis tagged protein fragments of mouse cdh23 and pcdh15 were previously described in [ , ]. .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707).

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: Paragraph title: Cloning and in vitro expression of backbone cyclized Crp4 variants ... Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth.

    Article Title: Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System *
    Article Snippet: Paragraph title: Expression Constructs ... The DNA encoding 1–76 amino acids of Rub1 was cloned into pTXB1 vector (NEB) using standard techniques, and the construct was verified by DNA sequencing.

    Transformation Assay:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: .. Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Transformed BL21(DE3) cells were induced with 0.3 mM IPTG for 3 h at 30 °C and transformed Origami2(DE3) cells with 0.1 mM IPTG for 20 h at 22 °C.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. Transformation of E. coli BL11 cells (New England Biolabs) was performed by mixing 25 μL cell suspension and 2 μL plasmid and incubating on ice for 30 mins.

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. This plasmid was transformed into T7 Express E. coli cells (New England Biolabs).

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: .. Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After selection on solid LB media containing ampicillin (100 µg/mL), individual colonies were used to inoculate 1 L of LB media containing ampicillin (100 µg/mL) and grown at 37°C to late log phase.

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: .. Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Transformed BL21(DE3) cells were induced with 0.3 mM IPTG for 4 h at 30 °C and transformed Origami2(DE3) cells with 0.1 mM IPTG for 20 h at 22 °C.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. Transformation of E. coli BL11 cells (New England Biolabs) was performed by mixing 25 μL cell suspension and 2 μL plasmid and incubating on ice for 30 mins.

    Ligation:

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: Paragraph title: Construction and purification of H97MeH CzrA using native chemical ligation ... The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Cell Culture:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The transformed cells were cultured at 310 K to late exponential phase in 5 l Luria–Bertani medium containing 50 µg ml−1 ampicillin and 34 µg ml−1 chloramphenicol.

    Generated:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: A DNA fragment encoding the cytoplasmic fragment of FlhB (Gene ID 1193710; amino-acid residues 213–350) was generated by PCR from genomic DNA of A. aeolicus as a template using the primers 5′-CAT ATG AAG ATA ATG ATG TCG AGA AGG GAA TTG-3′ and 5′-GCT CTT CCG CAG GCG TAA ACC TTT TTC TTT TTG AAC-3′ containing Nde I and Sap I restriction sites. .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD).

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: All mutants used in this work were generated using the QuikChange Lightning mutagenesis kit (Stratagene) and were verified by DNA sequencing. .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707).

    DNA Sequencing:

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: All mutants used in this work were generated using the QuikChange Lightning mutagenesis kit (Stratagene) and were verified by DNA sequencing. .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707).

    Article Title: Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System *
    Article Snippet: .. The DNA encoding 1–76 amino acids of Rub1 was cloned into pTXB1 vector (NEB) using standard techniques, and the construct was verified by DNA sequencing. .. Details of the cloning procedure and primer sequences are provided in the supplemental information.

    Sequencing:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    Article Title: BDNF pro-peptide regulates dendritic spines via caspase-3
    Article Snippet: .. DNA construct and purification of BDNF pro-peptide DNA sequence encoding the human BDNF pro-peptide (Val66 ) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between Nde I and Spe I sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. .. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S).

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: .. 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. This plasmid was transformed into T7 Express E. coli cells (New England Biolabs).

    Article Title: Brain-derived neurotrophic factor propeptide inhibits proliferation and induces apoptosis in C6 glioma cells
    Article Snippet: .. DNA construct and purification of the BDNF propeptide A DNA sequence that encodes for the human BDNF propeptide up to the furin cleavage site was inserted between the NdeI and SpeI sites of a pTXB1 vector (New England Biolab, Ipswich, Massachusetts, USA). .. Recombinant BDNF propeptide was expressed in BL21 Escherichia coli and purified using an IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) kit according to the manufacturer’s protocol (cat. #E6901S; New England Biolab).

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: .. The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion. .. The CzrA 1–95-intein fusion was expressed in E. coli BL21(DE3) and after sonication in Buffer C (25 mM Tris, 0.5 M NaCl, 2 mM TCEP, pH 8.0), was found to remain in the low speed lysis pellet.

    Sonication:

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Cells were lysed in 0.1 mM EDTA, 1 mM PMSF, 50 mM sodium phosphate, 250 mM sodium chloride buffer at pH 7.2 containing 5% glycerol by sonication.

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After incubating overnight at 16°C, cells were harvested by centrifugation, resuspended in 25 mL of 10 mM Tris-HCl, 500 mM NaCl buffer, pH 8.0 (Buffer A) and sonicated at 4°C.

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion. .. The CzrA 1–95-intein fusion was expressed in E. coli BL21(DE3) and after sonication in Buffer C (25 mM Tris, 0.5 M NaCl, 2 mM TCEP, pH 8.0), was found to remain in the low speed lysis pellet.

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Cells were lysed in 0.1 mM EDTA, 1 mM PMSF, 50 mM sodium phosphate, 250 mM sodium chloride buffer at pH 7.2 containing 5% glycerol by sonication.

    Affinity Purification:

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). .. Each intein tag contains a chitin-binding domain (CBD) for affinity purification of the fusion protein on a chitin resin.

    Recombinant:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    Article Title: BDNF pro-peptide regulates dendritic spines via caspase-3
    Article Snippet: DNA construct and purification of BDNF pro-peptide DNA sequence encoding the human BDNF pro-peptide (Val66 ) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between Nde I and Spe I sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. .. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S).

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: Paragraph title: Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation ... DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs).

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. Recombinant Rex (rRex) protein was purified from induced cell biomass according to protocols supplied with the IMPACT protein purification kit (New England Biolabs).

    Article Title: Brain-derived neurotrophic factor propeptide inhibits proliferation and induces apoptosis in C6 glioma cells
    Article Snippet: DNA construct and purification of the BDNF propeptide A DNA sequence that encodes for the human BDNF propeptide up to the furin cleavage site was inserted between the NdeI and SpeI sites of a pTXB1 vector (New England Biolab, Ipswich, Massachusetts, USA). .. Recombinant BDNF propeptide was expressed in BL21 Escherichia coli and purified using an IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) kit according to the manufacturer’s protocol (cat. #E6901S; New England Biolab).

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Mutagenesis:

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: Paragraph title: Cloning and mutagenesis ... Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707).

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. Plasmid DNA was isolated by miniprep and then sequenced (using T7short and T7 terminator primers) to confirm the mutation, and to confirm that D23N-Aβ1-29 was correctly aligned into the pTXB1 vector. .. Vector DNA was transformed into BL21DE3 or ER2566 cells and plated onto LB-amp plates.

    Isolation:

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. Plasmid DNA was isolated by miniprep and then sequenced (using T7short and T7 terminator primers) to confirm the mutation, and to confirm that D23N-Aβ1-29 was correctly aligned into the pTXB1 vector. .. Vector DNA was transformed into BL21DE3 or ER2566 cells and plated onto LB-amp plates.

    Purification:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    Article Title: BDNF pro-peptide regulates dendritic spines via caspase-3
    Article Snippet: .. DNA construct and purification of BDNF pro-peptide DNA sequence encoding the human BDNF pro-peptide (Val66 ) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between Nde I and Spe I sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. .. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S).

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: .. 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. This plasmid was transformed into T7 Express E. coli cells (New England Biolabs).

    Article Title: Brain-derived neurotrophic factor propeptide inhibits proliferation and induces apoptosis in C6 glioma cells
    Article Snippet: .. DNA construct and purification of the BDNF propeptide A DNA sequence that encodes for the human BDNF propeptide up to the furin cleavage site was inserted between the NdeI and SpeI sites of a pTXB1 vector (New England Biolab, Ipswich, Massachusetts, USA). .. Recombinant BDNF propeptide was expressed in BL21 Escherichia coli and purified using an IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) kit according to the manufacturer’s protocol (cat. #E6901S; New England Biolab).

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: .. Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After selection on solid LB media containing ampicillin (100 µg/mL), individual colonies were used to inoculate 1 L of LB media containing ampicillin (100 µg/mL) and grown at 37°C to late log phase.

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: Paragraph title: Construction and purification of H97MeH CzrA using native chemical ligation ... The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion.

    Protein Purification:

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: .. 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. This plasmid was transformed into T7 Express E. coli cells (New England Biolabs).

    Polymerase Chain Reaction:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: A DNA fragment encoding the cytoplasmic fragment of FlhB (Gene ID 1193710; amino-acid residues 213–350) was generated by PCR from genomic DNA of A. aeolicus as a template using the primers 5′-CAT ATG AAG ATA ATG ATG TCG AGA AGG GAA TTG-3′ and 5′-GCT CTT CCG CAG GCG TAA ACC TTT TTC TTT TTG AAC-3′ containing Nde I and Sap I restriction sites. .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD).

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). ..

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. As described above, a miniprep of DNA for the pTXB1 vector containing Aβ1-29 was used as a template for synthesis of the mutated strand by PCR. .. The ligation reaction was transformed into XL10-Gold Ultracompetent cells (Stratagene) and resulting colonies were screened on (LB) plates containing amp.

    IA:

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: .. Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Transformed BL21(DE3) cells were induced with 0.3 mM IPTG for 3 h at 30 °C and transformed Origami2(DE3) cells with 0.1 mM IPTG for 20 h at 22 °C.

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: .. Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Transformed BL21(DE3) cells were induced with 0.3 mM IPTG for 4 h at 30 °C and transformed Origami2(DE3) cells with 0.1 mM IPTG for 20 h at 22 °C.

    Plasmid Preparation:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    Article Title: BDNF pro-peptide regulates dendritic spines via caspase-3
    Article Snippet: .. DNA construct and purification of BDNF pro-peptide DNA sequence encoding the human BDNF pro-peptide (Val66 ) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between Nde I and Spe I sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. .. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S).

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: .. Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Transformed BL21(DE3) cells were induced with 0.3 mM IPTG for 3 h at 30 °C and transformed Origami2(DE3) cells with 0.1 mM IPTG for 20 h at 22 °C.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. DNA encoding Aβ1-29 was cloned into pTXB1 vector [ ], which utilizes an intein tag system. .. This vector was chosen based on its ability to generate a C-terminal thioester suitable for native chemical ligation after cleaving the intein tag using MESNA.

    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
    Article Snippet: .. 2.8 Rex Protein purification Rex protein was purified by first expressing the C. bescii rex coding sequence on plasmid pTXB1 (New England Biolabs part number N6707S, Ipswitch, MA) upstream of the gyrase intein and chitin‐binding domain (CBD), yielding plasmid pTSB1::rex (Table B). .. This plasmid was transformed into T7 Express E. coli cells (New England Biolabs).

    Article Title: Brain-derived neurotrophic factor propeptide inhibits proliferation and induces apoptosis in C6 glioma cells
    Article Snippet: .. DNA construct and purification of the BDNF propeptide A DNA sequence that encodes for the human BDNF propeptide up to the furin cleavage site was inserted between the NdeI and SpeI sites of a pTXB1 vector (New England Biolab, Ipswich, Massachusetts, USA). .. Recombinant BDNF propeptide was expressed in BL21 Escherichia coli and purified using an IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) kit according to the manufacturer’s protocol (cat. #E6901S; New England Biolab).

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: .. The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion. .. The CzrA 1–95-intein fusion was expressed in E. coli BL21(DE3) and after sonication in Buffer C (25 mM Tris, 0.5 M NaCl, 2 mM TCEP, pH 8.0), was found to remain in the low speed lysis pellet.

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins
    Article Snippet: .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). ..

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: .. Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth. .. Transformed BL21(DE3) cells were induced with 0.3 mM IPTG for 4 h at 30 °C and transformed Origami2(DE3) cells with 0.1 mM IPTG for 20 h at 22 °C.

    Article Title: Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System *
    Article Snippet: .. The DNA encoding 1–76 amino acids of Rub1 was cloned into pTXB1 vector (NEB) using standard techniques, and the construct was verified by DNA sequencing. .. Details of the cloning procedure and primer sequences are provided in the supplemental information.

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. As described above, a miniprep of DNA for the pTXB1 vector containing Aβ1-29 was used as a template for synthesis of the mutated strand by PCR. .. The ligation reaction was transformed into XL10-Gold Ultracompetent cells (Stratagene) and resulting colonies were screened on (LB) plates containing amp.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. Production recombinant tau fragments (tau1–415-Mxe-CBD) for ligation DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: .. Plasmid DNA was isolated by miniprep and then sequenced (using T7short and T7 terminator primers) to confirm the mutation, and to confirm that D23N-Aβ1-29 was correctly aligned into the pTXB1 vector. .. Vector DNA was transformed into BL21DE3 or ER2566 cells and plated onto LB-amp plates.

    Selection:

    Article Title: Fidelity Index Determination of DNA Methyltransferases
    Article Snippet: Cloning, expression and purification of methyltransferases The genes for all methyltransferases were cloned into pTXB1 (NEB #N6707) and transformed into E. coli strain T7 Express (NEB #C2566). .. After selection on solid LB media containing ampicillin (100 µg/mL), individual colonies were used to inoculate 1 L of LB media containing ampicillin (100 µg/mL) and grown at 37°C to late log phase.

    Agarose Gel Electrophoresis:

    Article Title: Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus
    Article Snippet: .. After confirming the sequence, the DNA fragment was digested from the vector with Nde I and Sap I, purified on agarose gel and cloned into the pTXB1 vector (New England BioLabs), generating an in-frame fusion with the intein and chitin-binding domain (CBD). .. The recombinant vector containing the gene for the FlhBc-intein-CBD fusion protein was transformed into Escherichia coli strain Rosetta (DE3) (Novagen).

    In Vitro:

    Article Title: Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system
    Article Snippet: Paragraph title: Cloning of RTD-C3 and RTD-C7 and in vitro production of RTD-1 ... Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding RTD-1 ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously., The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth.

    Article Title: Biosynthesis and antimicrobial evaluation of backbone-cyclized alpha-defensins#
    Article Snippet: Paragraph title: Cloning and in vitro expression of backbone cyclized Crp4 variants ... Synthetic DNA oligos (Integrated DNA technologies, Coralville, IA) encoding the different backbone cyclized Crp4 analogs ( ) were annealed and ligated into the pTXB1 vector (New England Biolabs, Ipswich, MA) using the NdeI and SapI restriction sites as described previously. ( , ) The resulting plasmids were transformed into either BL21(DE3) or Origami2(DE3) cells (EMD Chemicals, Gibbstown, NJ) and grown in LB broth.

    Concentration Assay:

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion. .. This pellet was then resuspended in Buffer C containing 7 M urea and refolded by stepwise decreasing the urea concentration in Buffer C. The resultant soluble fraction of CzrA 1–95-intein was cleaved with the addition of 100 mM -mercaptoethanesulfonate (MESNA) (Sigma, MO) with CzrA 1–95-thioester further purified on a C18 reverse phase column by running a 0–75% acetonitrile gradient in 0.1% TFA.

    Lysis:

    Article Title: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family
    Article Snippet: The DNA sequence encoding the N-terminal peptide (residue 1–95) was cloned into the pTXB1 vector (New England Biolabs) between NdeI and SpeI restriction sites in frame to a C-terminal intein fusion. .. The CzrA 1–95-intein fusion was expressed in E. coli BL21(DE3) and after sonication in Buffer C (25 mM Tris, 0.5 M NaCl, 2 mM TCEP, pH 8.0), was found to remain in the low speed lysis pellet.

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    New England Biolabs ptxb1 vector
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Ptxb1 Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Journal: Biopolymers

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    doi: 10.1002/bip.21391

    Figure Lengend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Article Snippet: As described above, a miniprep of DNA for the pTXB1 vector containing Aβ1-29 was used as a template for synthesis of the mutated strand by PCR.

    Techniques: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation