top10  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    MultiShot StripWell TOP10 Chemically Competent E. coli
    Description:
    MultiShot StripWell TOP10 chemically competent E. coli cells are cloning competent cells that packaged in a rack containing 12 strips of 8 tubes to increase productivity for medium-throughput bacterial transformations. TOP10 competent E. coli cells are ideal for high-efficiency cloning and plasmid propagation. They allow stable replication of high-copy number plasmids and are the same competent cells that come with many of our cloning kits. Versatile TOP10 cloning capabilities MultiShot FlexPlate TOP10 competent E. coli cells are the laboratory workhorse for chemical transformations as they are compatible with a broad spectrum of DNA types for transformation. The TOP10 strain is useful for many cloning applications and provides transformation efficiencies in the MultiShot FlexPlate format of >1 x 108 cfu/µg with control plasmid DNA. Key features of the MultiShot FlexPlate TOP10 Competent Cells include: • hsdR for efficient transformation of unmethylated DNA from PCR amplifications • mcrA for efficient transformation of methylated DNA from genomic preparations • lacZΔM15 for blue/white color screening of recombinant clones • endA1 for cleaner preparations of DNA and better results in downstream applications due to the elimination of non-specific digestion by endonuclease I • recA1 for reduced occurrence of non-specific recombination in cloned DNA MultiShot StripWell format—flexible for no waste Each StripWell of eight connected tubes is designed with the same single-tube, single-use features as our One Shot format tubes. This format allows for all steps of the transformation protocol, up to plating, to take place in the same tube, thereby saving time and preventing contamination. After addition of DNA, MultiShot StripWell competent cells can be transformed by heat-shock at 42°C using a water bath, all in the same tube. The StripWell tubes can be cut to permit transformations in a single tube, eliminating wasted aliquots of cells and avoiding freeze-thaws that can result in reduced transformation efficiency. Key features of the MultiShot StripWell format include: • Increased productivity—for medium throughput experimentation • Maximal flexibility—transform as few as 1 tube and as many as 96 from the StripWell rack • Familiar convenience—no aliquotting, add DNA directly to cells, heat shock, and recover in the same tube • Peace of mind—single-use transformation minimizes contamination Genotype: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara, leu)7697 galU galK rpsL (StrR) endA1 nupG Find the strain and format that you need We offer many other E. coli strains and formats of chemically competent cells and electrocompetent cells to help meet your specific transformation needs. If you require other high-throughput transformation options, choose from our collection of MultiShot formatted comp cells or email us for custom formatting options.
    Catalog Number:
    C409601
    Price:
    None
    Applications:
    Chemically Competent Cells for Cloning|Cloning|Transformation
    Size:
    1 rack
    Category:
    Competent Cells & Strains, Cloning Competent Cells
    Score:
    85
    Buy from Supplier
    Name:
    MultiShot TOP10 Chemically Competent E. coli
    Description:
    MultiShot TOP10 Chemically Competent E. coli provide transformation efficiencies of >1 x 108 cfu/µg control plasmid DNA, and are suited for high-throughput transformations. MultiShot TOP10 Chemically Competent cells are packaged in five 96-well microtiter plates (15 µL aliquots) to simplify high-throughput bacterial transformations. Benefits of MultiShot TOP10 cells:• Designed for high-throughput applications—microtiter plate format designed for automated high-throughput cloning• Compatible with genomic DNA—the mcrA mutation supports efficient transfection of methylated DNA• Permits rapid screening—the LacZΔM15 allele permits rapid blue/white screening of transfectants on plates containing X-Gal or Bluo-Gal• Efficient—the endA1 mutation helps reduce endonuclease activity, improving subsequent plasmid DNA yields• Reliable—the recA1 mutation helps reduce unwanted recombinationEasy to use for high-throughput transformationsMultiShot TOP10 Chemically Competent cells are genetically similar to the reliable DH10B strain, and are available in five 96-well plates to facilitate high-throughput transformation. After addition of DNA, MultiShot TOP10 Chemically Competent cells can be transformed by heat-shock at 42°C using a heat block or thermocycler. Using our TA Cloning and TOPO Cloning protocols, including controls, yields of 100–400 colonies per agar plate can be expected.Genotype: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara, leu)7697 galU galK rpsL (StrR) endA1 nupGFind the strain and format that you needWe also offer many other different strains and formats of chemically competent cells and electrocompetent cells to help meet your specific transformation needs. If you require other high-throughput transformation options, choose from our collection of MultiShot formatted comp cells or contact us for custom formatting options.
    Catalog Number:
    C40005
    Price:
    None
    Applications:
    Chemically Competent Cells for Cloning|Cloning|Transformation
    Size:
    5 plates
    Category:
    Competent Cells & Strains, Cloning Competent Cells
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher top10
    Induction kinetics of pvuIICR operon. ( A ) System used. To allow stable titration with C.PvuII, its positive feedback loop was broken by placing the pvuIIC gene under control of the arabinose-inducible P araBAD promoter (top), while fusing the lacZ reporter gene (β-galactosidase) to the promoter that normally controls pvuIIC and is regulated by C.PvuII (P pvuIICR , middle; including both P CR 1 and P CR 2). The strain background was E. coli <t>TOP10.</t> The tet gene (tetracycline resistance) is on the same plasmid as lacZ , and was used to normalize gene expression in some experiments. The C.PvuII binding sites are shown at the bottom, with ovals representing C.PvuII homodimers. Some experiments use a non-repressing variant, in which C-box 2B (bottom) is altered ( AG TC → GA TC). For references, see text. ( B ) C.PvuII levels. Results of C.PvuII measurements from western blots of cell extracts. C.PvuII was detected by a polyclonal primary antiserum, with final readout via luminescence densitometry. Means of triplicates, ±SE, are shown. The loading normalization was to BCCP, a naturally biotinylated E. coli protein. See Methods for details.
    MultiShot TOP10 Chemically Competent E. coli provide transformation efficiencies of >1 x 108 cfu/µg control plasmid DNA, and are suited for high-throughput transformations. MultiShot TOP10 Chemically Competent cells are packaged in five 96-well microtiter plates (15 µL aliquots) to simplify high-throughput bacterial transformations. Benefits of MultiShot TOP10 cells:• Designed for high-throughput applications—microtiter plate format designed for automated high-throughput cloning• Compatible with genomic DNA—the mcrA mutation supports efficient transfection of methylated DNA• Permits rapid screening—the LacZΔM15 allele permits rapid blue/white screening of transfectants on plates containing X-Gal or Bluo-Gal• Efficient—the endA1 mutation helps reduce endonuclease activity, improving subsequent plasmid DNA yields• Reliable—the recA1 mutation helps reduce unwanted recombinationEasy to use for high-throughput transformationsMultiShot TOP10 Chemically Competent cells are genetically similar to the reliable DH10B strain, and are available in five 96-well plates to facilitate high-throughput transformation. After addition of DNA, MultiShot TOP10 Chemically Competent cells can be transformed by heat-shock at 42°C using a heat block or thermocycler. Using our TA Cloning and TOPO Cloning protocols, including controls, yields of 100–400 colonies per agar plate can be expected.Genotype: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara, leu)7697 galU galK rpsL (StrR) endA1 nupGFind the strain and format that you needWe also offer many other different strains and formats of chemically competent cells and electrocompetent cells to help meet your specific transformation needs. If you require other high-throughput transformation options, choose from our collection of MultiShot formatted comp cells or contact us for custom formatting options.
    https://www.bioz.com/result/top10/product/Thermo Fisher
    Average 99 stars, based on 152 article reviews
    Price from $9.99 to $1999.99
    top10 - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "A bistable hysteretic switch in an activator-repressor regulated restriction-modification system"

    Article Title: A bistable hysteretic switch in an activator-repressor regulated restriction-modification system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt324

    Induction kinetics of pvuIICR operon. ( A ) System used. To allow stable titration with C.PvuII, its positive feedback loop was broken by placing the pvuIIC gene under control of the arabinose-inducible P araBAD promoter (top), while fusing the lacZ reporter gene (β-galactosidase) to the promoter that normally controls pvuIIC and is regulated by C.PvuII (P pvuIICR , middle; including both P CR 1 and P CR 2). The strain background was E. coli TOP10. The tet gene (tetracycline resistance) is on the same plasmid as lacZ , and was used to normalize gene expression in some experiments. The C.PvuII binding sites are shown at the bottom, with ovals representing C.PvuII homodimers. Some experiments use a non-repressing variant, in which C-box 2B (bottom) is altered ( AG TC → GA TC). For references, see text. ( B ) C.PvuII levels. Results of C.PvuII measurements from western blots of cell extracts. C.PvuII was detected by a polyclonal primary antiserum, with final readout via luminescence densitometry. Means of triplicates, ±SE, are shown. The loading normalization was to BCCP, a naturally biotinylated E. coli protein. See Methods for details.
    Figure Legend Snippet: Induction kinetics of pvuIICR operon. ( A ) System used. To allow stable titration with C.PvuII, its positive feedback loop was broken by placing the pvuIIC gene under control of the arabinose-inducible P araBAD promoter (top), while fusing the lacZ reporter gene (β-galactosidase) to the promoter that normally controls pvuIIC and is regulated by C.PvuII (P pvuIICR , middle; including both P CR 1 and P CR 2). The strain background was E. coli TOP10. The tet gene (tetracycline resistance) is on the same plasmid as lacZ , and was used to normalize gene expression in some experiments. The C.PvuII binding sites are shown at the bottom, with ovals representing C.PvuII homodimers. Some experiments use a non-repressing variant, in which C-box 2B (bottom) is altered ( AG TC → GA TC). For references, see text. ( B ) C.PvuII levels. Results of C.PvuII measurements from western blots of cell extracts. C.PvuII was detected by a polyclonal primary antiserum, with final readout via luminescence densitometry. Means of triplicates, ±SE, are shown. The loading normalization was to BCCP, a naturally biotinylated E. coli protein. See Methods for details.

    Techniques Used: Titration, Plasmid Preparation, Expressing, Binding Assay, Variant Assay, Western Blot

    2) Product Images from "Improvement of bacterial transformation efficiency using plasmid artificial modification"

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn884

    The transformation of Bifidobacterium was confirmed by plasmid isolation followed by agarose gel electrophoresis. Plasmids extracted from PAM host E. coli TOP10 harbouring pPAM1233–1283 (Lane 1) and from recombinant B. adolescentis ATCC15703 (Lane 2). Vector pKKT427 (Lane 3) and pPAM1233-1283 (Lane 4).
    Figure Legend Snippet: The transformation of Bifidobacterium was confirmed by plasmid isolation followed by agarose gel electrophoresis. Plasmids extracted from PAM host E. coli TOP10 harbouring pPAM1233–1283 (Lane 1) and from recombinant B. adolescentis ATCC15703 (Lane 2). Vector pKKT427 (Lane 3) and pPAM1233-1283 (Lane 4).

    Techniques Used: Transformation Assay, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Recombinant

    Comparison of PAM effects on transformation efficiencies. ( A–D ) Bifidobacterium adolescentis ATCC15703 was transformed by electroporation using the PAM method. The plasmid pKKT427 was prepared from E. coli TOP10 carrying pPAM1233-1283 ( A ), pPAM1233 ( B ), pPAM1283 ( C ) or without pPAM plasmid ( D ). An alkaline-SDS method using purification by agarose gel electrophoresis was used to isolate the PAM plasmids which were then introduced into B. adolescentis ATCC15703 by electroporation, as described previously ( 6 ). The electroporated samples were 100 times diluted in (A–C ) , but not in D. ( E ) Schematic presentation of transformation efficiencies. Plasmid pKKT427 was prepared from the PAM host (blue), B. longum 105-A (green) or B. adolescentis ATCC15703. The numbers beside arrows indicate transformation efficiencies (CFU/µg DNA).
    Figure Legend Snippet: Comparison of PAM effects on transformation efficiencies. ( A–D ) Bifidobacterium adolescentis ATCC15703 was transformed by electroporation using the PAM method. The plasmid pKKT427 was prepared from E. coli TOP10 carrying pPAM1233-1283 ( A ), pPAM1233 ( B ), pPAM1283 ( C ) or without pPAM plasmid ( D ). An alkaline-SDS method using purification by agarose gel electrophoresis was used to isolate the PAM plasmids which were then introduced into B. adolescentis ATCC15703 by electroporation, as described previously ( 6 ). The electroporated samples were 100 times diluted in (A–C ) , but not in D. ( E ) Schematic presentation of transformation efficiencies. Plasmid pKKT427 was prepared from the PAM host (blue), B. longum 105-A (green) or B. adolescentis ATCC15703. The numbers beside arrows indicate transformation efficiencies (CFU/µg DNA).

    Techniques Used: Transformation Assay, Electroporation, Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Electrophoresis

    3) Product Images from "AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach"

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137652

    AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli Top10 cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.
    Figure Legend Snippet: AQUA Cloning conditions. (a) The DNA fragment encoding the red fluorescent protein mCherry was PCR amplified with flanking extensions of 16, 24, or 32 bp of homologous sequence overlaps to an SV40 promoter-driven mammalian expression vector which was digested, or PCR amplified. AQUA Cloning was performed with pre-incubations on ice, at room temperature (RT), or at 50°C. DNA mixtures were transformed into chemically competent E . coli Top10 cells and colonies were obtained the next day. (b) Typical numbers of colony forming units (CFU) obtained from each condition. The highest number of CFU was derived from a pre-incubation at room temperature and with 32 bp of shared homology with PCR originating DNA fragments (arrow). The accuracies for each condition were determined by analytical colony PCR from eight selected clones and were extrapolated to the total number of CFU (grey). Abbreviations: P SV40 , simian virus 40 early promoter.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing, Expressing, Plasmid Preparation, Transformation Assay, Derivative Assay, Incubation

    AQUA Expression—combined cloning and protein expression. (a) Timeline for AQUA Expression. Cloning and production of recombinant protein in E . coli may be performed within 24 h starting with the PCR until the bacteria are harvested the next day. (b) A 3-DNA fragment cloning was performed by inserting the coding sequence for the red fluorescent protein mCherry into a bacterial T7 promoter-driven expression vector. The vector was split into two parts within the resistance gene for the antibiotic spectinomycin. Therefore, only correctly assembled fragments allow cell growth. (c) AQUA Expression in the expression strain BL21 (DE3) results in red colored bacteria due to mCherry protein production, while the TOP10 strain—lacking the required T7 RNA polymerase—remains colorless.
    Figure Legend Snippet: AQUA Expression—combined cloning and protein expression. (a) Timeline for AQUA Expression. Cloning and production of recombinant protein in E . coli may be performed within 24 h starting with the PCR until the bacteria are harvested the next day. (b) A 3-DNA fragment cloning was performed by inserting the coding sequence for the red fluorescent protein mCherry into a bacterial T7 promoter-driven expression vector. The vector was split into two parts within the resistance gene for the antibiotic spectinomycin. Therefore, only correctly assembled fragments allow cell growth. (c) AQUA Expression in the expression strain BL21 (DE3) results in red colored bacteria due to mCherry protein production, while the TOP10 strain—lacking the required T7 RNA polymerase—remains colorless.

    Techniques Used: Expressing, Clone Assay, Recombinant, Polymerase Chain Reaction, Sequencing, Plasmid Preparation

    AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.
    Figure Legend Snippet: AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.

    Techniques Used: Clone Assay, Produced, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Flow Cytometry, Generated, Amplification, Derivative Assay, Purification, Nucleic Acid Electrophoresis, Incubation, Transformation Assay, In Vivo

    4) Product Images from "Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes"

    Article Title: Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsw034

    Mapping the sequence determinants of -35 motifs in Ehrlichia chaffeensis genes. The β-galactosidase expression driven by E. chaffeensis promoters constructs containing point mutations at each of the six nucleotide positions of the -35 motifs of genes encoding p28-Omp14 (A) and p28-Omp19 (B) were measured in the CAG20177 strain of E. coli expressing E. chaffeensis σ 70 . The experiment included the no promoter (NP) and wild-type promoter (WT) controls. Each mutation is identified with a change of the nucleotide at each position to the modified nucleotide. β-galactosidase expression was presented relative to the respective wild-type promoters. The β-galactosidase expression driven by E. chaffeensis promoters constructs containing point mutations at each of the six nucleotide positions of the -35 motifs of genes encoding p28-Omp14 (C) and p28-Omp19 (D) also were measured in the TOP10 strain of E. coli expressing its native chromosomally expressed σ 70 with only the promoter plasmid pQF50K-p28-Omp14 or pQF50K-p28-Omp19 . The experiment also included the no promoter (NP) and wild-type promoter (WT) controls. Only four substititions in p28-Omp14 gene promoter and five substitions in p28-Omp19 gene promoter correlated well in altering the promoter activities when using σ 70 of E. chaffeensis and E. coli (within ∼10% variations); these mutations were identified in this figure with bold text.
    Figure Legend Snippet: Mapping the sequence determinants of -35 motifs in Ehrlichia chaffeensis genes. The β-galactosidase expression driven by E. chaffeensis promoters constructs containing point mutations at each of the six nucleotide positions of the -35 motifs of genes encoding p28-Omp14 (A) and p28-Omp19 (B) were measured in the CAG20177 strain of E. coli expressing E. chaffeensis σ 70 . The experiment included the no promoter (NP) and wild-type promoter (WT) controls. Each mutation is identified with a change of the nucleotide at each position to the modified nucleotide. β-galactosidase expression was presented relative to the respective wild-type promoters. The β-galactosidase expression driven by E. chaffeensis promoters constructs containing point mutations at each of the six nucleotide positions of the -35 motifs of genes encoding p28-Omp14 (C) and p28-Omp19 (D) also were measured in the TOP10 strain of E. coli expressing its native chromosomally expressed σ 70 with only the promoter plasmid pQF50K-p28-Omp14 or pQF50K-p28-Omp19 . The experiment also included the no promoter (NP) and wild-type promoter (WT) controls. Only four substititions in p28-Omp14 gene promoter and five substitions in p28-Omp19 gene promoter correlated well in altering the promoter activities when using σ 70 of E. chaffeensis and E. coli (within ∼10% variations); these mutations were identified in this figure with bold text.

    Techniques Used: Sequencing, Expressing, Construct, Mutagenesis, Modification, Plasmid Preparation

    5) Product Images from "Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system"

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1010

    Methylation status of plasmid isolated from cells expressing varied levels of WT M.PvuII or its variants. The complementary strands for the symmetrized C-box region of the PvuII R-M system are shown at the top. The upper strand specifies M.PvuII. The two changes that symmetrize the C-boxes are shown in red, along with the resultant changes in the MTase sequence (N4S/S10R). Substitution of the O L spacer, from CAT to CAA, would result in an additional change (M8L). Escherichia coli TOP10 cells carried two plasmids: pUC × 7PvuII (with seven PvuII sites) and plasmids expressing either WT pvuIIM (pBadMTwt-kan) or one of its variants (MTase N4S/S10R–pBadMT2-kan; MTase N4S/S10R/M8L–pBadMT2-kan) under the arabinose inducible promoter P BAD . After induction with a range of arabinose concentrations, cells were pelleted and plasmid DNA was isolated. The extent of methylation was assessed by digestion with PvuII restriction enzyme. Results ranged from full cleavage (no protection as in control lane 1; asterisk shows the position of highest bands) to no cleavage (complete methylation as in control lane 2). Digests were resolved on 1% agarose gel. Lanes 4 to 8 for each MTase represent equivalent increasing arabinose concentration from 0.01 to 0.2%. The topmost band in lanes 4–8 for each MTase is the pBAD plasmid linearized with XhoI prior to digestion with PvuII indicated by arrow; pUC × 7PvuII lacks a XhoI site. M lane shows DNA markers (1 kB Plus ladder; Invitrogen).
    Figure Legend Snippet: Methylation status of plasmid isolated from cells expressing varied levels of WT M.PvuII or its variants. The complementary strands for the symmetrized C-box region of the PvuII R-M system are shown at the top. The upper strand specifies M.PvuII. The two changes that symmetrize the C-boxes are shown in red, along with the resultant changes in the MTase sequence (N4S/S10R). Substitution of the O L spacer, from CAT to CAA, would result in an additional change (M8L). Escherichia coli TOP10 cells carried two plasmids: pUC × 7PvuII (with seven PvuII sites) and plasmids expressing either WT pvuIIM (pBadMTwt-kan) or one of its variants (MTase N4S/S10R–pBadMT2-kan; MTase N4S/S10R/M8L–pBadMT2-kan) under the arabinose inducible promoter P BAD . After induction with a range of arabinose concentrations, cells were pelleted and plasmid DNA was isolated. The extent of methylation was assessed by digestion with PvuII restriction enzyme. Results ranged from full cleavage (no protection as in control lane 1; asterisk shows the position of highest bands) to no cleavage (complete methylation as in control lane 2). Digests were resolved on 1% agarose gel. Lanes 4 to 8 for each MTase represent equivalent increasing arabinose concentration from 0.01 to 0.2%. The topmost band in lanes 4–8 for each MTase is the pBAD plasmid linearized with XhoI prior to digestion with PvuII indicated by arrow; pUC × 7PvuII lacks a XhoI site. M lane shows DNA markers (1 kB Plus ladder; Invitrogen).

    Techniques Used: Methylation, Plasmid Preparation, Isolation, Expressing, Sequencing, Cellular Antioxidant Activity Assay, Agarose Gel Electrophoresis, Concentration Assay

    In vivo effects of C-box O L spacer variants on temporal expression of C.PvuII. ( A ) Diagram of experiment. Four E. coli TOP10’ cultures carrying plasmids with different O L spacers fused to a cat (chloramphenicol acetyltransferase) promotorless reporter gene were infected with recombinant M13pvuIIwt phage at MOI = 15. We used three spacer variants tested previously in this study: WT-Sym (CAT/CAA; pDK178; closed circles), CAA/CAA (pIM22; open circles) and nonrepressing WT-Sym (box 2B mutated, pWWWR; gray circles) as a control. The first two variants have identical, symmetrized C-boxes sequences, and differ only at a single nt in O R . A plasmid with no C-box sequence (pKK-238; triangles) was also tested and gave only background levels of cat expression (shown in Figure S6). ( B ) Growth was monitored at OD 600 nm before and after phage addition. ( C ) CAT production over infection time for two independently performed experiments was measured by sensitive colorimetric assay based on ELISA sandwich method (see ‘Materials and methods’ section). CAT production data were normalized to the value for each strain prior to infection. Curves were fitted and statistical analysis was performed using the SAS package (SAS Institute Inc., Cary, NC). The analysis of covariance indicated significant difference of slopes over time with P
    Figure Legend Snippet: In vivo effects of C-box O L spacer variants on temporal expression of C.PvuII. ( A ) Diagram of experiment. Four E. coli TOP10’ cultures carrying plasmids with different O L spacers fused to a cat (chloramphenicol acetyltransferase) promotorless reporter gene were infected with recombinant M13pvuIIwt phage at MOI = 15. We used three spacer variants tested previously in this study: WT-Sym (CAT/CAA; pDK178; closed circles), CAA/CAA (pIM22; open circles) and nonrepressing WT-Sym (box 2B mutated, pWWWR; gray circles) as a control. The first two variants have identical, symmetrized C-boxes sequences, and differ only at a single nt in O R . A plasmid with no C-box sequence (pKK-238; triangles) was also tested and gave only background levels of cat expression (shown in Figure S6). ( B ) Growth was monitored at OD 600 nm before and after phage addition. ( C ) CAT production over infection time for two independently performed experiments was measured by sensitive colorimetric assay based on ELISA sandwich method (see ‘Materials and methods’ section). CAT production data were normalized to the value for each strain prior to infection. Curves were fitted and statistical analysis was performed using the SAS package (SAS Institute Inc., Cary, NC). The analysis of covariance indicated significant difference of slopes over time with P

    Techniques Used: In Vivo, Expressing, Infection, Recombinant, Cellular Antioxidant Activity Assay, Plasmid Preparation, Sequencing, Colorimetric Assay, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Optimization of production of recombinant human growth hormone in Escherichia coli"

    Article Title: Optimization of production of recombinant human growth hormone in Escherichia coli

    Journal: Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences

    doi:

    Dot blot analysis of E. coli strains expressed rhGH in LB and 4YT mediums. A1: GH 1 mgr/ml , A2: GH 100 μgr/ml , A3: GH 10 μgr/ml , A4: GH 1 μgr/ ml , A5: GH 100 ngr/ml , A6: PBS, A7: control – with out loading, A8: Top10 none transformed supernatant, A9: Top10 none transformed cell lysate, A10: Top10 transformed LB T0 supernatant, A11: Top10 transformed LB T0 cell lysate, B1: Top10 transformed LB T5 supernatant, B2: Top10 transformed LB T5 supernatant 1/10 diluted, B3: Top10 transformed LB T5 cell lysate, B4: Top10 transformed LB T5 cell lysate 1/10 diluted, B5: Top10 transformed 4YT T0 supernatant, B6: Top10 transformed 4YT T0 cell lysate, B7: Top10 transformed 4YT T5 supernatant, B8: Top10 transformed 4YT T5 supernatant 1/10 diluted, B9: Top10 transformed 4YT T5 cell lysate, B10: Top10 transformed 4YT T5 cell lysate 1/10 diluted, B11 to C11: Top10 repeat 2, D1 to E1: Top10 repeat 3. E2 to F4: repeat 1 of XL1-blue, F5 to G5: repeat 2, G6 to H6: repeat 3. H7 to I9: repeat 1 of JM109, I10 to J10: repeat 2 and J11 to K11: repeat 3.
    Figure Legend Snippet: Dot blot analysis of E. coli strains expressed rhGH in LB and 4YT mediums. A1: GH 1 mgr/ml , A2: GH 100 μgr/ml , A3: GH 10 μgr/ml , A4: GH 1 μgr/ ml , A5: GH 100 ngr/ml , A6: PBS, A7: control – with out loading, A8: Top10 none transformed supernatant, A9: Top10 none transformed cell lysate, A10: Top10 transformed LB T0 supernatant, A11: Top10 transformed LB T0 cell lysate, B1: Top10 transformed LB T5 supernatant, B2: Top10 transformed LB T5 supernatant 1/10 diluted, B3: Top10 transformed LB T5 cell lysate, B4: Top10 transformed LB T5 cell lysate 1/10 diluted, B5: Top10 transformed 4YT T0 supernatant, B6: Top10 transformed 4YT T0 cell lysate, B7: Top10 transformed 4YT T5 supernatant, B8: Top10 transformed 4YT T5 supernatant 1/10 diluted, B9: Top10 transformed 4YT T5 cell lysate, B10: Top10 transformed 4YT T5 cell lysate 1/10 diluted, B11 to C11: Top10 repeat 2, D1 to E1: Top10 repeat 3. E2 to F4: repeat 1 of XL1-blue, F5 to G5: repeat 2, G6 to H6: repeat 3. H7 to I9: repeat 1 of JM109, I10 to J10: repeat 2 and J11 to K11: repeat 3.

    Techniques Used: Dot Blot, Transformation Assay

    Comparison of transformation efficiency between different E.coli strains (A) Top10 (B) XL1-blue and (C) JM109
    Figure Legend Snippet: Comparison of transformation efficiency between different E.coli strains (A) Top10 (B) XL1-blue and (C) JM109

    Techniques Used: Transformation Assay

    7) Product Images from "Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate"

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate

    Journal:

    doi: 10.1016/j.ymben.2009.01.008

    The isoprenoid biosynthetic pathway in E. coli (via deoxyxylulose phosphate (DXP) pathway) and in archaea (via mevalonate pathway) for ether lipid biosynthesis. Indicated genes were cloned and expressed in E. coli TOP10.
    Figure Legend Snippet: The isoprenoid biosynthetic pathway in E. coli (via deoxyxylulose phosphate (DXP) pathway) and in archaea (via mevalonate pathway) for ether lipid biosynthesis. Indicated genes were cloned and expressed in E. coli TOP10.

    Techniques Used: Clone Assay

    Western blot analysis with an anti-His antibody confirmed expression of His-tagged G1P dehydrogenase (39.4 kDa) and GGGP synthase (30.4 kDa) in E. coli . Lane 1: Crude cell lysate of E. coli TOP10/pDL11/pDL6. Lane 2: Affinity chromatography fraction of E. coli TOP10/pDL6 lysate containing His-tagged GGGP synthase. Lane 3: Affinity chromatography fraction of E. coli TOP10/pDL11 lysate containing His-tagged G1P dehydrogenase.
    Figure Legend Snippet: Western blot analysis with an anti-His antibody confirmed expression of His-tagged G1P dehydrogenase (39.4 kDa) and GGGP synthase (30.4 kDa) in E. coli . Lane 1: Crude cell lysate of E. coli TOP10/pDL11/pDL6. Lane 2: Affinity chromatography fraction of E. coli TOP10/pDL6 lysate containing His-tagged GGGP synthase. Lane 3: Affinity chromatography fraction of E. coli TOP10/pDL11 lysate containing His-tagged G1P dehydrogenase.

    Techniques Used: Western Blot, Expressing, Affinity Chromatography

    8) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M111.306118

    Stalled intermediates interact with BamA and BamD during OM translocation. TOP10 cells expressing empty vector ( EV ), wild-type Pet, PetSB, Pet48aa, Pet1HA, and Pet1HA-FLAG were harvested, spheroplasted, and lysed. Proteins were co-purified with anti-Pet
    Figure Legend Snippet: Stalled intermediates interact with BamA and BamD during OM translocation. TOP10 cells expressing empty vector ( EV ), wild-type Pet, PetSB, Pet48aa, Pet1HA, and Pet1HA-FLAG were harvested, spheroplasted, and lysed. Proteins were co-purified with anti-Pet

    Techniques Used: Translocation Assay, Expressing, Plasmid Preparation, Positron Emission Tomography, Purification

    Stalled intermediates possess a hairpin conformation that can be detected using fluorescence microscopy. TOP10 cells expressing empty vector ( EV ), PetSB, PetSB-FLAG, PetL4HA, Pet1HA, and Pet1HA-FLAG were collected 1 h after induction with 0.02% arabinose
    Figure Legend Snippet: Stalled intermediates possess a hairpin conformation that can be detected using fluorescence microscopy. TOP10 cells expressing empty vector ( EV ), PetSB, PetSB-FLAG, PetL4HA, Pet1HA, and Pet1HA-FLAG were collected 1 h after induction with 0.02% arabinose

    Techniques Used: Fluorescence, Microscopy, Expressing, Plasmid Preparation

    A rigid linker interferes with OM translocation. A , SDS-PAGE analysis of TCA-precipitated culture supernatant fractions harvested after growth of TOP10 and TOP10 Δ dsbA expressing Pet, Pet1HA ( 1HA ), Pet2HA ( 2HA ), and Pet3HA ( 3HA ). Intramolecular
    Figure Legend Snippet: A rigid linker interferes with OM translocation. A , SDS-PAGE analysis of TCA-precipitated culture supernatant fractions harvested after growth of TOP10 and TOP10 Δ dsbA expressing Pet, Pet1HA ( 1HA ), Pet2HA ( 2HA ), and Pet3HA ( 3HA ). Intramolecular

    Techniques Used: Translocation Assay, SDS Page, Expressing, Positron Emission Tomography

    9) Product Images from "Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli"

    Article Title: Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-1053-3_3

    Phenotypic diversity a. Diversity in individual colony fluorescence intensity. E. coli Top10 cells were transformed with the pGFPuv library obtained by four rounds of direct-plating mutagenesis and grown under carbenicillin selection (the antibiotic marker for the plasmid). The majority of these colonies represent single plasmid transformations. Colonies were imaged using a UVP bioanalyzer illuminated at 302nm using a SYBR filter with an emission cutoff between 517-570 nm. b. Flow cytometry analysis. Colonies shown in panel b were washed with LB and grown to an OD600 between 0.7-0.9 for optimal GFP fluorescence ( see ). Next, cells were diluted in sheath solution ( see ) to an event rate of less than 100 cells per second passing through the detector of the BD Influx cytometer. The GFP fluorescence was analyzed using a 531/40 optical filter and excited by a 488nm laser. This data represents the fluorescence emission of single cells in a cell culture population. In addition to the library, we also show two controls: cells expressing WT pGFPuv plasmid, and untransformed cells. These are labeled directly on the figure.
    Figure Legend Snippet: Phenotypic diversity a. Diversity in individual colony fluorescence intensity. E. coli Top10 cells were transformed with the pGFPuv library obtained by four rounds of direct-plating mutagenesis and grown under carbenicillin selection (the antibiotic marker for the plasmid). The majority of these colonies represent single plasmid transformations. Colonies were imaged using a UVP bioanalyzer illuminated at 302nm using a SYBR filter with an emission cutoff between 517-570 nm. b. Flow cytometry analysis. Colonies shown in panel b were washed with LB and grown to an OD600 between 0.7-0.9 for optimal GFP fluorescence ( see ). Next, cells were diluted in sheath solution ( see ) to an event rate of less than 100 cells per second passing through the detector of the BD Influx cytometer. The GFP fluorescence was analyzed using a 531/40 optical filter and excited by a 488nm laser. This data represents the fluorescence emission of single cells in a cell culture population. In addition to the library, we also show two controls: cells expressing WT pGFPuv plasmid, and untransformed cells. These are labeled directly on the figure.

    Techniques Used: Fluorescence, Transformation Assay, Mutagenesis, Selection, Marker, Plasmid Preparation, Flow Cytometry, Cytometry, Cell Culture, Expressing, Labeling

    Related Articles

    Clone Assay:

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach
    Article Snippet: For additional guidance see “Troubleshooting guide for AQUA Cloning”. .. Except when indicated otherwise, AQUA Cloning was performed in the TOP10 (Invitrogen) E . coli strain which was prepared for competency using the following protocol: 20 mL LB-medium were inoculated in a 100 mL sterile Erlenmeyer flask and were incubated overnight at 37°C, 300 r.p.m. .. Next day, the pre-culture was used to inoculate four 500 mL sterile Erlenmeyer flasks, each containing 250 mL LB-medium which were incubated at 37°C, 250 r.p.m, until OD600 = 0.5–0.6 was reached.

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system
    Article Snippet: MC1061 [araD139 Δ(ara, leu )7697, ΔlacX74 , galU , galK , hsdR , strA ] ( ) is able to transport arabinose but is deficient in its metabolism; it was used as the host for in vivo titrations with C.PvuII. .. TOP10 (Invitrogen; mcr A Δmrr-hsdRMS-mcrBC φ80lacZM15 lacX74 recA1 araD139 (ara-leu )7697 galU galK rpsL (StrR ) endA1 nupG’) was used for all other purposes including cloning steps and CAT assay. .. [Top10 with F′ (lacIq , Tn10 (TetR )] was used as the host strain for M13 phage infections.

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification
    Article Snippet: The bacterial strains, B. adolescentis ATCC15703 was obtained from the American Type Culture Collection. .. An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression. .. Bifidobacterium adolescentis ATCC15703 was grown anaerobically at 37°C in MRS medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 0.02% L -cysteine (Nacalai Tesque, Kyoto, Japan) and 0.34% l -ascorbic acid sodium salt (Nacalai Tesque).

    Article Title: A bistable hysteretic switch in an activator-repressor regulated restriction-modification system
    Article Snippet: The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq . .. The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq .

    Article Title: Fermentation of lactose to ethanol in cheese whey permeate and concentrated permeate by engineered Escherichia coli
    Article Snippet: Our biocatalyst could efficiently ferment two dairy waste streams, derived from an existing valorization chain, without nutritional supplements, providing promising results towards the green, sustainable and economically attractive conversion of such waste into a biofuel. .. TOP10 (Invitrogen) E. coli were used for cloning according to manufacturer’s instructions. .. Bacterial hosts for ethanol production are described in Table .

    Article Title: Half-life measurements of chemical inducers for recombinant gene expression
    Article Snippet: Detailed description about plasmid construction, as well as full DNA sequences, can be found in the individual parts web pages of the Registry [ ]. .. TOP10 (Invitrogen) E. coli strain was used as a host for cloning. .. MG1655-Z1 E. coli strain [ ] was used as the final host for the biosensing plasmids, as its genome encodes constitutive LacI and TetR over-expression cassettes.

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The PCR products of Kaiso regulatory region and PU.1 promoter region were ligated to multiple cloning site of the pGL4.10 [luc2 ] Vector (Promega)(Supplementary Fig. ). .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter.

    Article Title: Revisiting the role of phospholipases C in virulence and the lifecycle ofMycobacterium tuberculosis
    Article Snippet: In conclusion, our study calls into question the impact of PLCs on virulence of M.tuberculosis , and provides new hints on putative alternative functions of PLCs in M. tuberculosis . .. Escherichia coli DH10B and Top10 (Invitrogen) strains, used for cloning procedures, were grown on LB agar medium and/or LB broth. .. M. smegmatis mc2 155 and M. tuberculosis strains were obtained from stock held at the Institut Pasteur.

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
    Article Snippet: The designed artificial genes were prepared by an artificial-gene synthesis (Bioneer Corp., Daejon, Korea) and then cloned into T-vector (Promega, Madison, WI, USA), finally resulting in the pMMP319 and pMMP320 ( and ). .. Plasmids were introduced into Top10 (Invitrogen, Carlsbad, CA, USA), E. coli competent cells, by heat-shock with RbCl2 treatment.

    Centrifugation:

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach
    Article Snippet: Except when indicated otherwise, AQUA Cloning was performed in the TOP10 (Invitrogen) E . coli strain which was prepared for competency using the following protocol: 20 mL LB-medium were inoculated in a 100 mL sterile Erlenmeyer flask and were incubated overnight at 37°C, 300 r.p.m. .. Next day, the pre-culture was used to inoculate four 500 mL sterile Erlenmeyer flasks, each containing 250 mL LB-medium which were incubated at 37°C, 250 r.p.m, until OD600 = 0.5–0.6 was reached.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8. .. The membrane vesicles were solubilized by adding DDM to a final concentration of 40 mM.

    Amplification:

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate
    Article Snippet: A. fulgidus genomic DNA was isolated using a purification kit (Promega) for use as PCR gene templates for amplification of egsA , GGGPS, and DGGGPS. .. The E. coli strains DH5α (Stratagene) and TOP10 (Invitrogen) were used for plasmid constructions, and E. coli TOP10 was used for all gene expression studies and for DGGGP production.

    Article Title: A bistable hysteretic switch in an activator-repressor regulated restriction-modification system
    Article Snippet: The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq . .. The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq .

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The PU.1 promoter region in Ctse gene was amplified with 5′-primer with Kpn I site (5′-GGGGG GGTACC GGACCCTCATTCACTTTTGC-3′) and 3′-primer with Xho I site (5′-GGGGG CTCGAG GAGACAGGGCCTTACCTGTG-3′). .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter.

    Acetylene Reduction Assay:

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system
    Article Snippet: MC1061 [araD139 Δ(ara, leu )7697, ΔlacX74 , galU , galK , hsdR , strA ] ( ) is able to transport arabinose but is deficient in its metabolism; it was used as the host for in vivo titrations with C.PvuII. .. TOP10 (Invitrogen; mcr A Δmrr-hsdRMS-mcrBC φ80lacZM15 lacX74 recA1 araD139 (ara-leu )7697 galU galK rpsL (StrR ) endA1 nupG’) was used for all other purposes including cloning steps and CAT assay. .. [Top10 with F′ (lacIq , Tn10 (TetR )] was used as the host strain for M13 phage infections.

    Construct:

    Article Title: Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes
    Article Snippet: In view of the lack of a transformation system in E. chaffeensis and in other related tick-borne intracellular rickettsial pathogens, the assessment of Ehrlichia transcriptional machinery in the surrogate E. coli system along with the validation experiments carried out by in vitro transcription assays offer innovative means in studying gene expression in E. chaffeensis and other important intracellular rickettsial pathogens belonging to the Anaplasmataceae family. .. Escherichia coli strains used in this study were TOP10 (Invitrogen Technologies, Carlsbad, CA), BL21(DE3) (Novagen, San Diego, CA) and CAG20177., Several plasmid constructs used in this study were obtained from a commercial source or modified from one or more of the existing plasmids. .. They include pET32a (Novagen) and the derivatives of pSAKT32, pQF50K and pMT504.

    Incubation:

    Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach
    Article Snippet: For additional guidance see “Troubleshooting guide for AQUA Cloning”. .. Except when indicated otherwise, AQUA Cloning was performed in the TOP10 (Invitrogen) E . coli strain which was prepared for competency using the following protocol: 20 mL LB-medium were inoculated in a 100 mL sterile Erlenmeyer flask and were incubated overnight at 37°C, 300 r.p.m. .. Next day, the pre-culture was used to inoculate four 500 mL sterile Erlenmeyer flasks, each containing 250 mL LB-medium which were incubated at 37°C, 250 r.p.m, until OD600 = 0.5–0.6 was reached.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: Insoluble matter was removed by ultracentrifugation, and the soluble supernatant was incubated with TALON-affinity resin (Takara Bio Inc.) overnight at 4°C. .. TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8.

    Luciferase:

    Article Title: Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells
    Article Snippet: This lysogen contains a firefly luciferase expression cassette under the transcriptional control of the cytomegalovirus immediate early promoter. .. Bacteriophage λD1180(luc) lysogens were prepared in Top10 (Invitrogen) Escherichia coli ( E. coli) hosts and transformed with a wild-type gpD-containing plasmid, pTrc-gpD , to produce luciferase-expressing bacteriophage lambda [λ(luc)]. .. Induction of the lysogen and purification of the λ(luc) particles was performed as described ( ). λ(luc) titers were measured by plaque forming units (PFU) on LE392 E. coli cells.

    Activity Assay:

    Article Title: Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli
    Article Snippet: The JS200-EP and JS200-WT strains, as well as the LF Pol I-bearing plasmid, its map and sequence are available by request through the Addgene plasmid repository ( ). .. Readout strain, JS200-WT or (for complementation) a strain lacking the specific activity that is being evolved such as Top10 (Invitrogen, Grand Island, NY, USA). .. LB Agar and LB broth were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and prepared according to vendor specifications.

    Expressing:

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification
    Article Snippet: The bacterial strains, B. adolescentis ATCC15703 was obtained from the American Type Culture Collection. .. An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression. .. Bifidobacterium adolescentis ATCC15703 was grown anaerobically at 37°C in MRS medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 0.02% L -cysteine (Nacalai Tesque, Kyoto, Japan) and 0.34% l -ascorbic acid sodium salt (Nacalai Tesque).

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate
    Article Snippet: A. fulgidus genomic DNA was isolated using a purification kit (Promega) for use as PCR gene templates for amplification of egsA , GGGPS, and DGGGPS. .. The E. coli strains DH5α (Stratagene) and TOP10 (Invitrogen) were used for plasmid constructions, and E. coli TOP10 was used for all gene expression studies and for DGGGP production. .. For routine cultivation, E. coli was grown in Luria-Bertani medium at 37 °C on a rotary shaker at 200 rpm.

    Article Title: Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli
    Article Snippet: E. coli JS200 strains ( recA718 polA12 (ts) uvrA155 trpE65 lon-11 sulA): JS200-WT, i.e. JS200 cells expressing wild type (wt) Pol I; and JS200-EP, i.e. JS200 expressing error prone (EP) Pol I. .. Readout strain, JS200-WT or (for complementation) a strain lacking the specific activity that is being evolved such as Top10 (Invitrogen, Grand Island, NY, USA).

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The PCR products of Kaiso regulatory region and PU.1 promoter region were ligated to multiple cloning site of the pGL4.10 [luc2 ] Vector (Promega)(Supplementary Fig. ). .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter. .. Thus, CGCG and CGGG sites were not methylated in TOP10 competent cells.

    Article Title: Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells
    Article Snippet: Bacteriophage λD1180(luc) lysogens were prepared in Top10 (Invitrogen) Escherichia coli ( E. coli) hosts and transformed with a wild-type gpD-containing plasmid, pTrc-gpD , to produce luciferase-expressing bacteriophage lambda [λ(luc)]. .. Bacteriophage λD1180(luc) lysogens were prepared in Top10 (Invitrogen) Escherichia coli ( E. coli) hosts and transformed with a wild-type gpD-containing plasmid, pTrc-gpD , to produce luciferase-expressing bacteriophage lambda [λ(luc)].

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: Paragraph title: Protein expression and purification ... TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8.

    Modification:

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification
    Article Snippet: A Bifidobactrium-E. coli shuttle vector, pKKT427 , was modified from a pBRASTA101 replicon ( ). .. An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression.

    Article Title: Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes
    Article Snippet: In view of the lack of a transformation system in E. chaffeensis and in other related tick-borne intracellular rickettsial pathogens, the assessment of Ehrlichia transcriptional machinery in the surrogate E. coli system along with the validation experiments carried out by in vitro transcription assays offer innovative means in studying gene expression in E. chaffeensis and other important intracellular rickettsial pathogens belonging to the Anaplasmataceae family. .. Escherichia coli strains used in this study were TOP10 (Invitrogen Technologies, Carlsbad, CA), BL21(DE3) (Novagen, San Diego, CA) and CAG20177., Several plasmid constructs used in this study were obtained from a commercial source or modified from one or more of the existing plasmids. .. They include pET32a (Novagen) and the derivatives of pSAKT32, pQF50K and pMT504.

    Transformation Assay:

    Article Title: Half-life measurements of chemical inducers for recombinant gene expression
    Article Snippet: TOP10 (Invitrogen) E. coli strain was used as a host for cloning. .. MG1655-Z1 E. coli strain [ ] was used as the final host for the biosensing plasmids, as its genome encodes constitutive LacI and TetR over-expression cassettes.

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The PCR products of Kaiso regulatory region and PU.1 promoter region were ligated to multiple cloning site of the pGL4.10 [luc2 ] Vector (Promega)(Supplementary Fig. ). .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter. .. Thus, CGCG and CGGG sites were not methylated in TOP10 competent cells.

    Article Title: Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells
    Article Snippet: This lysogen contains a firefly luciferase expression cassette under the transcriptional control of the cytomegalovirus immediate early promoter. .. Bacteriophage λD1180(luc) lysogens were prepared in Top10 (Invitrogen) Escherichia coli ( E. coli) hosts and transformed with a wild-type gpD-containing plasmid, pTrc-gpD , to produce luciferase-expressing bacteriophage lambda [λ(luc)]. .. Induction of the lysogen and purification of the λ(luc) particles was performed as described ( ). λ(luc) titers were measured by plaque forming units (PFU) on LE392 E. coli cells.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: VcINDY was eluted, and the affinity tag removed, by incubating the protein/resin mixture with 10 μg/ml trypsin for 1 hr at 4°C. vSGLT was expressed and purified as detailed previously ( ). .. TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8. .. Expression was induced by adding 0.66 mM L-arabinose.

    Article Title: Initial assembly steps of a translocase for folded proteins
    Article Snippet: The RR-precursor proteins TorA-mCherry, TorA-MalE and pSufI were synthesized by in vitro transcription/translation using the plasmids indicated in . .. Cell extracts were prepared from E. coli strain SL119 (ref. ) or Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of RR-precursors . .. These cell extracts were used to synthesize radioactively labelled proteins from plasmid DNA by coupled transcription/translation in 50 μl reactions .

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
    Article Snippet: Paragraph title: 5.4. Plasmid Construction for the Secretion of MP-V1 and Transformation into a General Host Strain ... Plasmids were introduced into Top10 (Invitrogen, Carlsbad, CA, USA), E. coli competent cells, by heat-shock with RbCl2 treatment.

    Translocation Assay:

    Article Title: Initial assembly steps of a translocase for folded proteins
    Article Snippet: Cell extracts were prepared from E. coli strain SL119 (ref. ) or Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of RR-precursors . .. Cell extracts were prepared from E. coli strain SL119 (ref. ) or Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of RR-precursors .

    Derivative Assay:

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
    Article Snippet: The collected OmpA SS nucleotide sequence was designed to connect directly to the translational start site derived from the Ptrc promoter, and the MP-V1 was also designed to directly connect OmpA SS . .. Plasmids were introduced into Top10 (Invitrogen, Carlsbad, CA, USA), E. coli competent cells, by heat-shock with RbCl2 treatment.

    Low Copy Number:

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification
    Article Snippet: pBAD33 ( ) was used as low copy number cloning vector. .. An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression.

    Cell Culture:

    Article Title: Fermentation of lactose to ethanol in cheese whey permeate and concentrated permeate by engineered Escherichia coli
    Article Snippet: TOP10 (Invitrogen) E. coli were used for cloning according to manufacturer’s instructions. .. TOP10 (Invitrogen) E. coli were used for cloning according to manufacturer’s instructions.

    Article Title: Revisiting the role of phospholipases C in virulence and the lifecycle ofMycobacterium tuberculosis
    Article Snippet: Escherichia coli DH10B and Top10 (Invitrogen) strains, used for cloning procedures, were grown on LB agar medium and/or LB broth. .. The M. tuberculosis MT103 strain and the corresponding Myc2509ΔPLC mutant strain were a gift of Prof. Gicquel, Institut Pasteur.

    Generated:

    Article Title:
    Article Snippet: A polyclonal rabbit antiserum generated toward the Pet passenger domain ( ) and BamA and BamD ( ) have been previously described. .. The Escherichia coli strains used in this study were TOP10 (Invitrogen) and BW25113 dsbA :: kan ( ).

    other:

    Article Title: A 1-phytase type III effector interferes with plant hormone signaling
    Article Snippet: Bacterial strains and growth conditions Escherichia coli BL21(DE3) (Agilent Technologies Inc., Santa Clara, USA) and TOP10 (Life Technologies GmbH, Darmstadt, Germany) were cultivated at 37 °C in LB (lysogeny broth) medium , A. tumefaciens GV3101 and derivatives at 30 °C in YEB (yeast extract broth) and Xcv strain 85-103 and derivatives on NYG (nutrient yeast glycerol) agar plates supplemented with appropriate antibiotics.

    Sequencing:

    Article Title: Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes
    Article Snippet: Escherichia coli strains used in this study were TOP10 (Invitrogen Technologies, Carlsbad, CA), BL21(DE3) (Novagen, San Diego, CA) and CAG20177., Several plasmid constructs used in this study were obtained from a commercial source or modified from one or more of the existing plasmids. .. The plasmid pSAKT32 containing a p15A origin of replication and an ampicillin resistance gene has E. coli rpoH gene under the control of IPTG inducible Plac promoter.

    Article Title: Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli
    Article Snippet: The JS200-EP and JS200-WT strains, as well as the LF Pol I-bearing plasmid, its map and sequence are available by request through the Addgene plasmid repository ( ). .. Readout strain, JS200-WT or (for complementation) a strain lacking the specific activity that is being evolved such as Top10 (Invitrogen, Grand Island, NY, USA).

    Article Title: Cleavage of a putative metal permease in Chlamydia trachomatis yields an iron-dependent transcriptional repressor
    Article Snippet: Secondary structures were predicted using the Phyre2 Server ( ). .. All transformations were made into Top10 (Invitrogen) E. coli , and vectors were verified by sequencing (GATC). .. Expression of recombinant YtgR was induced from the pET-YtgR plasmid in BL21* (DE3) E. coli .

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
    Article Snippet: The collected OmpA SS nucleotide sequence was designed to connect directly to the translational start site derived from the Ptrc promoter, and the MP-V1 was also designed to directly connect OmpA SS . .. Plasmids were introduced into Top10 (Invitrogen, Carlsbad, CA, USA), E. coli competent cells, by heat-shock with RbCl2 treatment.

    Recombinant:

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system
    Article Snippet: TOP10 (Invitrogen; mcr A Δmrr-hsdRMS-mcrBC φ80lacZM15 lacX74 recA1 araD139 (ara-leu )7697 galU galK rpsL (StrR ) endA1 nupG’) was used for all other purposes including cloning steps and CAT assay. .. TOP10 (Invitrogen; mcr A Δmrr-hsdRMS-mcrBC φ80lacZM15 lacX74 recA1 araD139 (ara-leu )7697 galU galK rpsL (StrR ) endA1 nupG’) was used for all other purposes including cloning steps and CAT assay.

    Article Title: Half-life measurements of chemical inducers for recombinant gene expression
    Article Snippet: Figure describes the recombinant E. coli bearing the biosensors. .. TOP10 (Invitrogen) E. coli strain was used as a host for cloning.

    In Vivo:

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system
    Article Snippet: MC1061 [araD139 Δ(ara, leu )7697, ΔlacX74 , galU , galK , hsdR , strA ] ( ) is able to transport arabinose but is deficient in its metabolism; it was used as the host for in vivo titrations with C.PvuII. .. TOP10 (Invitrogen; mcr A Δmrr-hsdRMS-mcrBC φ80lacZM15 lacX74 recA1 araD139 (ara-leu )7697 galU galK rpsL (StrR ) endA1 nupG’) was used for all other purposes including cloning steps and CAT assay.

    Methylation:

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter. .. Thus, CGCG and CGGG sites were not methylated in TOP10 competent cells.

    Mutagenesis:

    Article Title: Revisiting the role of phospholipases C in virulence and the lifecycle ofMycobacterium tuberculosis
    Article Snippet: Escherichia coli DH10B and Top10 (Invitrogen) strains, used for cloning procedures, were grown on LB agar medium and/or LB broth. .. Escherichia coli DH10B and Top10 (Invitrogen) strains, used for cloning procedures, were grown on LB agar medium and/or LB broth.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: VcINDY was eluted, and the affinity tag removed, by incubating the protein/resin mixture with 10 μg/ml trypsin for 1 hr at 4°C. vSGLT was expressed and purified as detailed previously ( ). .. TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8. .. Expression was induced by adding 0.66 mM L-arabinose.

    Isolation:

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate
    Article Snippet: A. fulgidus genomic DNA was isolated using a purification kit (Promega) for use as PCR gene templates for amplification of egsA , GGGPS, and DGGGPS. .. The E. coli strains DH5α (Stratagene) and TOP10 (Invitrogen) were used for plasmid constructions, and E. coli TOP10 was used for all gene expression studies and for DGGGP production.

    Article Title: A bistable hysteretic switch in an activator-repressor regulated restriction-modification system
    Article Snippet: The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq . .. The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq .

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The Kaiso regulatory region in Ctse gene was amplified from B6 or MRL mice genomic DNAs isolated from splenic CD4+ T cells by using 5′-primer with Xho I restriction site (5′-GGGGGG CTCGAG GGACATCCTTCTAGGAAGCG-3′), and 3′-primer with Hind III restriction site (5′-GGGGGG AAGCTT GGTGTGTGTGTGTCTGATACTG-3′), and TaKaRa Taq Hot Start Version. .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: The lysate was clarified and membrane vesicles were isolated by ultracentrifugation. .. TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8.

    Microscopy:

    Article Title: Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage
    Article Snippet: E . coli DH5Δlac (BR2846) and Top10 (Invitrogen, Carlsbad, CA) were used for standard plasmid manipulations and propagation. .. E . coli DH5Δlac (BR2846) and Top10 (Invitrogen, Carlsbad, CA) were used for standard plasmid manipulations and propagation.

    Purification:

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate
    Article Snippet: A. fulgidus genomic DNA was isolated using a purification kit (Promega) for use as PCR gene templates for amplification of egsA , GGGPS, and DGGGPS. .. The E. coli strains DH5α (Stratagene) and TOP10 (Invitrogen) were used for plasmid constructions, and E. coli TOP10 was used for all gene expression studies and for DGGGP production.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: Paragraph title: Protein expression and purification ... TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8.

    Polymerase Chain Reaction:

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate
    Article Snippet: A. fulgidus genomic DNA was isolated using a purification kit (Promega) for use as PCR gene templates for amplification of egsA , GGGPS, and DGGGPS. .. The E. coli strains DH5α (Stratagene) and TOP10 (Invitrogen) were used for plasmid constructions, and E. coli TOP10 was used for all gene expression studies and for DGGGP production.

    Article Title: A bistable hysteretic switch in an activator-repressor regulated restriction-modification system
    Article Snippet: The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq . .. The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq .

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The PCR products of Kaiso regulatory region and PU.1 promoter region were ligated to multiple cloning site of the pGL4.10 [luc2 ] Vector (Promega)(Supplementary Fig. ). .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter.

    Positron Emission Tomography:

    Article Title:
    Article Snippet: A polyclonal rabbit antiserum generated toward the Pet passenger domain ( ) and BamA and BamD ( ) have been previously described. .. The Escherichia coli strains used in this study were TOP10 (Invitrogen) and BW25113 dsbA :: kan ( ).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system
    Article Snippet: MC1061 [araD139 Δ(ara, leu )7697, ΔlacX74 , galU , galK , hsdR , strA ] ( ) is able to transport arabinose but is deficient in its metabolism; it was used as the host for in vivo titrations with C.PvuII. .. TOP10 (Invitrogen; mcr A Δmrr-hsdRMS-mcrBC φ80lacZM15 lacX74 recA1 araD139 (ara-leu )7697 galU galK rpsL (StrR ) endA1 nupG’) was used for all other purposes including cloning steps and CAT assay. .. [Top10 with F′ (lacIq , Tn10 (TetR )] was used as the host strain for M13 phage infections.

    Mouse Assay:

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The Kaiso regulatory region in Ctse gene was amplified from B6 or MRL mice genomic DNAs isolated from splenic CD4+ T cells by using 5′-primer with Xho I restriction site (5′-GGGGGG CTCGAG GGACATCCTTCTAGGAAGCG-3′), and 3′-primer with Hind III restriction site (5′-GGGGGG AAGCTT GGTGTGTGTGTGTCTGATACTG-3′), and TaKaRa Taq Hot Start Version. .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter.

    Plasmid Preparation:

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification
    Article Snippet: The pKKT427, 3.9 kb vector, carried a spectinomycin resistance gene, a multi-cloning site and two replication origins including repB from B. longum and ColEI ori ( ). .. An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression.

    Article Title: Optimization of production of recombinant human growth hormone in Escherichia coli
    Article Snippet: Paragraph title: Bacterial Strains and Plasmid ... The E. coli host strains used in this study were TOP10 (Invitrogen, USA), XL1-blue and JM109 (Cinagen, Iran).

    Article Title: Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate
    Article Snippet: A. fulgidus genomic DNA was isolated using a purification kit (Promega) for use as PCR gene templates for amplification of egsA , GGGPS, and DGGGPS. .. The E. coli strains DH5α (Stratagene) and TOP10 (Invitrogen) were used for plasmid constructions, and E. coli TOP10 was used for all gene expression studies and for DGGGP production. .. For routine cultivation, E. coli was grown in Luria-Bertani medium at 37 °C on a rotary shaker at 200 rpm.

    Article Title: A bistable hysteretic switch in an activator-repressor regulated restriction-modification system
    Article Snippet: The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq . .. The genotypes of EPI300 (the CopyControl™ s train; Epicentre) and TOP10 (Invitrogen) are both F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔ M15 ΔlacX 74 recA 1 endA 1 araD 139 Δ(ara, leu) 7697 galU galK λ- rpsL (StrR ) nupG tonA . φ80lacZΔM15 contains the entire lac operon (though with part of lacZ deleted), including lacIq .

    Article Title: Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes
    Article Snippet: In view of the lack of a transformation system in E. chaffeensis and in other related tick-borne intracellular rickettsial pathogens, the assessment of Ehrlichia transcriptional machinery in the surrogate E. coli system along with the validation experiments carried out by in vitro transcription assays offer innovative means in studying gene expression in E. chaffeensis and other important intracellular rickettsial pathogens belonging to the Anaplasmataceae family. .. Escherichia coli strains used in this study were TOP10 (Invitrogen Technologies, Carlsbad, CA), BL21(DE3) (Novagen, San Diego, CA) and CAG20177., Several plasmid constructs used in this study were obtained from a commercial source or modified from one or more of the existing plasmids. .. They include pET32a (Novagen) and the derivatives of pSAKT32, pQF50K and pMT504.

    Article Title: Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli
    Article Snippet: The JS200-EP and JS200-WT strains, as well as the LF Pol I-bearing plasmid, its map and sequence are available by request through the Addgene plasmid repository ( ). .. Readout strain, JS200-WT or (for complementation) a strain lacking the specific activity that is being evolved such as Top10 (Invitrogen, Grand Island, NY, USA).

    Article Title: Half-life measurements of chemical inducers for recombinant gene expression
    Article Snippet: Detailed description about plasmid construction, as well as full DNA sequences, can be found in the individual parts web pages of the Registry [ ]. .. TOP10 (Invitrogen) E. coli strain was used as a host for cloning.

    Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells
    Article Snippet: The PCR products of Kaiso regulatory region and PU.1 promoter region were ligated to multiple cloning site of the pGL4.10 [luc2 ] Vector (Promega)(Supplementary Fig. ). .. Plasmids were transformed in TOP10 (Invitrogen) expressing Dam and Dcm methylases, which methylate GATC in the former, and CCAGG and CCTGG in the latter.

    Article Title: Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells
    Article Snippet: This lysogen contains a firefly luciferase expression cassette under the transcriptional control of the cytomegalovirus immediate early promoter. .. Bacteriophage λD1180(luc) lysogens were prepared in Top10 (Invitrogen) Escherichia coli ( E. coli) hosts and transformed with a wild-type gpD-containing plasmid, pTrc-gpD , to produce luciferase-expressing bacteriophage lambda [λ(luc)]. .. Induction of the lysogen and purification of the λ(luc) particles was performed as described ( ). λ(luc) titers were measured by plaque forming units (PFU) on LE392 E. coli cells.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: VcINDY was eluted, and the affinity tag removed, by incubating the protein/resin mixture with 10 μg/ml trypsin for 1 hr at 4°C. vSGLT was expressed and purified as detailed previously ( ). .. TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8. .. Expression was induced by adding 0.66 mM L-arabinose.

    Article Title: Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage
    Article Snippet: Bacterial strains, plasmids and primers are listed in – Tables, respectively. .. E . coli DH5Δlac (BR2846) and Top10 (Invitrogen, Carlsbad, CA) were used for standard plasmid manipulations and propagation. .. V . cholerae strains used were derivatives of CVC1121, a hapR + derivative of the sequenced strain N16961.

    Article Title: Initial assembly steps of a translocase for folded proteins
    Article Snippet: The RR-precursor proteins TorA-mCherry, TorA-MalE and pSufI were synthesized by in vitro transcription/translation using the plasmids indicated in . .. Cell extracts were prepared from E. coli strain SL119 (ref. ) or Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of RR-precursors . .. These cell extracts were used to synthesize radioactively labelled proteins from plasmid DNA by coupled transcription/translation in 50 μl reactions .

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
    Article Snippet: Paragraph title: 5.4. Plasmid Construction for the Secretion of MP-V1 and Transformation into a General Host Strain ... Plasmids were introduced into Top10 (Invitrogen, Carlsbad, CA, USA), E. coli competent cells, by heat-shock with RbCl2 treatment.

    Negative Control:

    Article Title: Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
    Article Snippet: The OmpA SS was fused directly to the Ptrc promoter without MP-V1 , and was designed to be used as a negative control. .. Plasmids were introduced into Top10 (Invitrogen, Carlsbad, CA, USA), E. coli competent cells, by heat-shock with RbCl2 treatment.

    Selection:

    Article Title: Revisiting the role of phospholipases C in virulence and the lifecycle ofMycobacterium tuberculosis
    Article Snippet: Escherichia coli DH10B and Top10 (Invitrogen) strains, used for cloning procedures, were grown on LB agar medium and/or LB broth. .. Mycobacterial strains were cultured in Middlebrook 7H9 broth supplemented with ADC (Difco) and 0.05% Tween 80 or on Middlebrook 7H11 medium supplemented with OADC (Difco).

    In Vitro:

    Article Title: Initial assembly steps of a translocase for folded proteins
    Article Snippet: Paragraph title: In vitro reactions ... Cell extracts were prepared from E. coli strain SL119 (ref. ) or Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of RR-precursors .

    Produced:

    Article Title:
    Article Snippet: Anti-HA tag antibody produced in rabbits, alkaline phosphatase-conjugated goat anti-rabbit antibodies, and the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolylphosphate were obtained from Sigma-Aldrich. .. The Escherichia coli strains used in this study were TOP10 (Invitrogen) and BW25113 dsbA :: kan ( ).

    Concentration Assay:

    Article Title: Fermentation of lactose to ethanol in cheese whey permeate and concentrated permeate by engineered Escherichia coli
    Article Snippet: TOP10 (Invitrogen) E. coli were used for cloning according to manufacturer’s instructions. .. TOP10 (Invitrogen) E. coli were used for cloning according to manufacturer’s instructions.

    Article Title: A general method for determining secondary active transporter substrate stoichiometry
    Article Snippet: Membrane vesicles were then resuspended in Purification Buffer (PB) containing 50 mM Tris-HCl, pH 8, 100 mM NaCl and 5% (vol/vol) glycerol and solubilized by adding n-dodecyl-β-D-maltoside (DDM; Anatrace) to a final concentration of 20 mM. .. TOP10 (Life Technologies Carlsbad, CA) cells, transformed with a pBAD vector containing the vSGLT gene in-frame with a C-terminal Histidine tag and the mutation A423C , were grown at 37°C in TB supplemented with 100 μg/ml ampicillin to an A600 of 1.8.

    Marker:

    Article Title: Half-life measurements of chemical inducers for recombinant gene expression
    Article Snippet: They have been assembled into the pSB3K3 medium-copy plasmid (with kanamycin-resistance marker and p15A replication origin) by using the BioBrick™ Standard Assembly procedure [ ] and conventional molecular biology techniques [ ]. .. TOP10 (Invitrogen) E. coli strain was used as a host for cloning.

    Staining:

    Article Title: Improvement of bacterial transformation efficiency using plasmid artificial modification
    Article Snippet: The bacterial strains, B. adolescentis ATCC15703 was obtained from the American Type Culture Collection. .. An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression. .. Bifidobacterium adolescentis ATCC15703 was grown anaerobically at 37°C in MRS medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 0.02% L -cysteine (Nacalai Tesque, Kyoto, Japan) and 0.34% l -ascorbic acid sodium salt (Nacalai Tesque).

    Synthesized:

    Article Title: Initial assembly steps of a translocase for folded proteins
    Article Snippet: The RR-precursor proteins TorA-mCherry, TorA-MalE and pSufI were synthesized by in vitro transcription/translation using the plasmids indicated in . .. Cell extracts were prepared from E. coli strain SL119 (ref. ) or Top10 (Invitrogen) transformed with plasmid pSup-BpaRS-6TRN(D286R) to express amber stop codon mutants of RR-precursors .

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/top10 escherichia coli/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    top10 escherichia coli - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher pentr d topo cloning kit with one shot top10 chemically competent e coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Pentr D Topo Cloning Kit With One Shot Top10 Chemically Competent E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pentr d topo cloning kit with one shot top10 chemically competent e coli/product/Thermo Fisher
    Average 94 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    pentr d topo cloning kit with one shot top10 chemically competent e coli - by Bioz Stars, 2020-01
    94/100 stars
      Buy from Supplier

    93
    Thermo Fisher topo ta cloning kit for subcloning with one shot top10 chemically competent e coli cells
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Topo Ta Cloning Kit For Subcloning With One Shot Top10 Chemically Competent E Coli Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/topo ta cloning kit for subcloning with one shot top10 chemically competent e coli cells/product/Thermo Fisher
    Average 93 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    topo ta cloning kit for subcloning with one shot top10 chemically competent e coli cells - by Bioz Stars, 2020-01
    93/100 stars
      Buy from Supplier

    77
    Thermo Fisher e coli top10 carrying vector pcr4 topo
    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli <t>TOP10</t> cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector <t>pCR4-TOPO,</t> pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.
    E Coli Top10 Carrying Vector Pcr4 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli top10 carrying vector pcr4 topo/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli top10 carrying vector pcr4 topo - by Bioz Stars, 2020-01
    77/100 stars
      Buy from Supplier

    Image Search Results


    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Clone Assay, Plasmid Preparation

    Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Derivative Assay, Plasmid Preparation, Clone Assay