Structured Review

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(A) Expression of the PvpA protein in E. coli under T7 promoter control. E. coli <t>DH5αMCR</t> cells harboring the recombinant plasmid pKPV.2 were subjected to selective induction of the T7 promoter. An SDS-PAGE autoradiograph of [ 35 S]methionine-labeled proteins expressed in E. coli (left panel) or a Western blot analysis of the same proteins as depicted in the left panel with MAb 1E5 (right panel) is shown. Cells in both panels were used without induction (lanes 1), with induction of the T7 promoter (lanes 2), or with induction in the presence of rifampin (lanes 3). A labeled arrow indicates the recombinant PvpA 55-kDa product expressed in E. coli . (B) Amino acid sequence and structural features of the PvpA protein from M. gallisepticum strain R. Amino acid residues are numbered on the right. A broken line marks the putative PvpA signal peptide. Labeled arrows show the position and direction of two directly repeated amino acid sequences (DR-1 and DR-2). A repeated motif consisting of four amino acids, PRPX (X is Met, Gln, or Asn), which appears 14 times within the PvpA C-terminal region is highlighted by boldface. The fourth codon, within a tract of five glutamine residues and in which a nonsense mutation has occurred, is marked by an arrowhead. Sixty proline residues within the C-terminal region are marked by asterisks.
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1) Product Images from "Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein"

Article Title: Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein

Journal: Infection and Immunity

doi:

(A) Expression of the PvpA protein in E. coli under T7 promoter control. E. coli DH5αMCR cells harboring the recombinant plasmid pKPV.2 were subjected to selective induction of the T7 promoter. An SDS-PAGE autoradiograph of [ 35 S]methionine-labeled proteins expressed in E. coli (left panel) or a Western blot analysis of the same proteins as depicted in the left panel with MAb 1E5 (right panel) is shown. Cells in both panels were used without induction (lanes 1), with induction of the T7 promoter (lanes 2), or with induction in the presence of rifampin (lanes 3). A labeled arrow indicates the recombinant PvpA 55-kDa product expressed in E. coli . (B) Amino acid sequence and structural features of the PvpA protein from M. gallisepticum strain R. Amino acid residues are numbered on the right. A broken line marks the putative PvpA signal peptide. Labeled arrows show the position and direction of two directly repeated amino acid sequences (DR-1 and DR-2). A repeated motif consisting of four amino acids, PRPX (X is Met, Gln, or Asn), which appears 14 times within the PvpA C-terminal region is highlighted by boldface. The fourth codon, within a tract of five glutamine residues and in which a nonsense mutation has occurred, is marked by an arrowhead. Sixty proline residues within the C-terminal region are marked by asterisks.
Figure Legend Snippet: (A) Expression of the PvpA protein in E. coli under T7 promoter control. E. coli DH5αMCR cells harboring the recombinant plasmid pKPV.2 were subjected to selective induction of the T7 promoter. An SDS-PAGE autoradiograph of [ 35 S]methionine-labeled proteins expressed in E. coli (left panel) or a Western blot analysis of the same proteins as depicted in the left panel with MAb 1E5 (right panel) is shown. Cells in both panels were used without induction (lanes 1), with induction of the T7 promoter (lanes 2), or with induction in the presence of rifampin (lanes 3). A labeled arrow indicates the recombinant PvpA 55-kDa product expressed in E. coli . (B) Amino acid sequence and structural features of the PvpA protein from M. gallisepticum strain R. Amino acid residues are numbered on the right. A broken line marks the putative PvpA signal peptide. Labeled arrows show the position and direction of two directly repeated amino acid sequences (DR-1 and DR-2). A repeated motif consisting of four amino acids, PRPX (X is Met, Gln, or Asn), which appears 14 times within the PvpA C-terminal region is highlighted by boldface. The fourth codon, within a tract of five glutamine residues and in which a nonsense mutation has occurred, is marked by an arrowhead. Sixty proline residues within the C-terminal region are marked by asterisks.

Techniques Used: Expressing, Recombinant, Plasmid Preparation, SDS Page, Autoradiography, Labeling, Western Blot, Sequencing, Mutagenesis

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Clone Assay:

Article Title: Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein
Article Snippet: The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.) and Y1090 (Promega, Madison, Wis.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
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Amplification:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: The region, including 206 bp of upstream and 106 bp of downstream sequence, was amplified with ClCdtABC-F and ClCdtABC-R (Table ) and initially cloned into pCR2.1 TOPO. .. Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen).

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.). .. Two oligonucleotides, 5′-CTGCTTAGTTGAGTGTTGTTCC-3′ and 5′-CCTGGGTAACAGATGCAA-3′, containing Hin dIII restriction sites at their ends, were used for PCR amplification and cloning of the vspO gene ( ).

In Vitro:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen). .. Colonies that appeared on 20 mg/ml of chloramphenicol medium were tested for CDT activity in the in vitro CDT assay.

Plasmid Preparation:

Article Title: Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein
Article Snippet: The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.) and Y1090 (Promega, Madison, Wis.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
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Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity
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Article Title: The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features
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Mutagenesis:

Article Title: Mutations in Flavobacterium johnsoniae secDF Result in Defects in Gliding Motility and Chitin Utilization
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Isolation:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
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Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: The geographic origin and site of isolation of M. bovis PG45 were previously described ( ). .. The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.).

Low Copy Number:

Article Title: Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity
Article Snippet: The E. coli strains used in this study were DH5αMCR (Gibco-BRL, Gaithersburg, MD), XL-1 blue MRF′ (Promega, Madison, WI), and Na+ /H+ antiporter-deficient KNabc (Δ nhaA Δ nhaB Δ chaA ) ( ). .. The plasmids used for gene cloning were low copy number pMW118 (Nippon Kayaku, Tokyo, Japan) and high expression vector pKK223–3 (Amersham Pharmacia Biotech, Piscataway, NJ).

Microscopy:

Article Title: Mutations in Flavobacterium johnsoniae secDF Result in Defects in Gliding Motility and Chitin Utilization
Article Snippet: The Escherichia coli strains used were DH5αMCR (Invitrogen) and HB101 ( ). .. Wild-type and mutant cells of F. johnsoniae were examined for movement over glass and agar surfaces by phase-contrast microscopy as previously described ( ).

Purification:

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: Clonal isolate 182, expressing a 75-kDa VspC product, was isolate and purified as previously described ( ). .. The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.).

Sequencing:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: The region, including 206 bp of upstream and 106 bp of downstream sequence, was amplified with ClCdtABC-F and ClCdtABC-R (Table ) and initially cloned into pCR2.1 TOPO. .. Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen).

Article Title: The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features
Article Snippet: The sequence analysis of the vsp locus was done on clonal isolate 6 of the M. bovis type strain PG45, which has the phenotype VspA+ VspB+ . .. The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.), KW251, and LE392 (Promega, Madison, Wis.).

Modification:

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: All clonal isolates were propagated at 37°C in a modified standard mycoplasma broth medium as previously described ( ). .. The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.).

Activity Assay:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen). .. Colonies that appeared on 20 mg/ml of chloramphenicol medium were tested for CDT activity in the in vitro CDT assay.

Construct:

Article Title: Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein
Article Snippet: The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.) and Y1090 (Promega, Madison, Wis.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: .. Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen). .. The construct was isolated from E. coli and electroporated into C. jejuni EF and 99/68, using a method adapted from that of Wassenaar et al. ( ).

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features
Article Snippet: The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.), KW251, and LE392 (Promega, Madison, Wis.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.) or in pGEM-7Z (Promega).

Expressing:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: Complementation of CDT-negative strains was achieved by the introduction of the Campylobacter shuttle vector pUOA18 , expressing a 2.4-kb region from C. jejuni 11168 containing the cdt genes ( ). .. Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen).

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: Clonal isolate 182, expressing a 75-kDa VspC product, was isolate and purified as previously described ( ). .. The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.).

Article Title: Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity
Article Snippet: The E. coli strains used in this study were DH5αMCR (Gibco-BRL, Gaithersburg, MD), XL-1 blue MRF′ (Promega, Madison, WI), and Na+ /H+ antiporter-deficient KNabc (Δ nhaA Δ nhaB Δ chaA ) ( ). .. The plasmids used for gene cloning were low copy number pMW118 (Nippon Kayaku, Tokyo, Japan) and high expression vector pKK223–3 (Amersham Pharmacia Biotech, Piscataway, NJ).

Polymerase Chain Reaction:

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.). .. Recombinant plasmid pKO35 , carrying the vspO gene, was constructed by cloning the PCR-amplified vspO gene from M. bovis PG45 clonal isolate 7 into the plasmid vector pBS.

Transformation Assay:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: .. Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen). .. The construct was isolated from E. coli and electroporated into C. jejuni EF and 99/68, using a method adapted from that of Wassenaar et al. ( ).

Recombinant:

Article Title: Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein
Article Snippet: The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.) and Y1090 (Promega, Madison, Wis.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: Iron Starvation of Bordetella avium Stimulates Expression of Five Outer Membrane Proteins and Regulates a Gene Involved in Acquiring Iron from Serum
Article Snippet: .. Escherichia coli DH5αF′tet (Life Technologies, Inc., Gaithersburg, Md.) and DH5αmcr (Life Technologies, Inc.) were used as hosts for recombinant plasmids. ..

Article Title: Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen
Article Snippet: The Escherichia coli strain used was DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.).

Article Title: The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features
Article Snippet: The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.), KW251, and LE392 (Promega, Madison, Wis.). .. Recombinant clones were constructed in the plasmid vector pBluescript II KS(+) (Stratagene, La Jolla, Calif.) or in pGEM-7Z (Promega).

Derivative Assay:

Article Title: Mutations in Flavobacterium johnsoniae gldF and gldG Disrupt Gliding Motility and Interfere with Membrane Localization of GldA
Article Snippet: F. johnsoniae UW101 (ATCC 17061) was the wild-type strain used in these studies, and all mutants were derived from this strain. .. The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies), S17-1 , and BW19851 ( ).

Article Title: Flavobacterium johnsoniae GldJ Is a Lipoprotein That Is Required for Gliding Motility
Article Snippet: F. johnsoniae UW101 (from the F. johnsoniae type strain ATCC 17061) was the wild type used in this study, and all mutants were derived from this strain ( ). .. The Escherichia coli strains used were DH5αMCR (Gibco BRL Life Technologies), S17-1 , and TOP10 (Invitrogen).

Article Title: Mutations in Flavobacterium johnsoniae secDF Result in Defects in Gliding Motility and Chitin Utilization
Article Snippet: F. johnsoniae MM101 (a derivative of F. johnsoniae ATCC 17061) ( ) was the wild-type strain used in this study, and all mutants were derived from this strain. .. The Escherichia coli strains used were DH5αMCR (Invitrogen) and HB101 ( ).

Article Title: Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA
Article Snippet: F. johnsoniae UW101 (ATCC 17061) was the wild-type strain used in these studies and all mutants were derived from this strain. .. The Escherichia coli strains used were DH5αMCR (GIBCO/BRL), HB101(13), and S17-1 ( ).

Gel Purification:

Article Title: Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens
Article Snippet: .. Following BamHI digestion and gel purification, the insert was ligated into dephosphorylated, BamHI-digested pUOA18, to make the construct pCDT, and transformed into DH5αMCR (Invitrogen). .. The construct was isolated from E. coli and electroporated into C. jejuni EF and 99/68, using a method adapted from that of Wassenaar et al. ( ).