top10 Thermo Fisher Search Results


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  • 99
    Thermo Fisher top10 electrocomp kit
    Top10 Electrocomp Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher top10
    The transformation of Bifidobacterium was confirmed by plasmid isolation followed by agarose gel electrophoresis. Plasmids extracted from PAM host E. coli <t>TOP10</t> harbouring pPAM1233–1283 (Lane 1) and from recombinant B. adolescentis ATCC15703 (Lane 2). Vector pKKT427 (Lane 3) and pPAM1233-1283 (Lane 4).
    Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher coli top10 f
    The transformation of Bifidobacterium was confirmed by plasmid isolation followed by agarose gel electrophoresis. Plasmids extracted from PAM host E. coli <t>TOP10</t> harbouring pPAM1233–1283 (Lane 1) and from recombinant B. adolescentis ATCC15703 (Lane 2). Vector pKKT427 (Lane 3) and pPAM1233-1283 (Lane 4).
    Coli Top10 F, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher competent top10
    Measured RPU values for promoters assembled in the same plasmid in <t>TOP10</t> and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
    Competent Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher ecoli top10
    Measured RPU values for promoters assembled in the same plasmid in <t>TOP10</t> and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
    Ecoli Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher one shot top10
    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 <t>Top10</t> 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.
    One Shot Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher top10 electrocompetent
    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 <t>Top10</t> 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.
    Top10 Electrocompetent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher bacterial top10 cells
    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 <t>Top10</t> 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.
    Bacterial Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher electrocomp top10 cells
    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 <t>Top10</t> 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.
    Electrocomp Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher top10 host cells
    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 <t>Top10</t> 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.
    Top10 Host Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher growth media top10
    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 <t>Top10</t> 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.
    Growth Media Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher chemically competent top10 cells
    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 <t>Top10</t> 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.
    Chemically Competent Top10 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher oneshot top10 competent cells
    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 <t>Top10</t> 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.
    Oneshot Top10 Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher top10 supercompetent cells
    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 <t>Top10</t> 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.
    Top10 Supercompetent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher top10 thermocompetent cells
    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 <t>Top10</t> 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.
    Top10 Thermocompetent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher oneshot top10 electrocomp cells
    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 <t>Top10</t> 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.
    Oneshot Top10 Electrocomp Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher top10 dh5α competent cells
    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 <t>Top10</t> 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.
    Top10 Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher oneshot top10 kit
    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 <t>Top10</t> 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.
    Oneshot Top10 Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher strain top10
    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 <t>Top10</t> 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.
    Strain Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher e coli lysogen host
    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 <t>Top10</t> 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.
    E Coli Lysogen Host, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher competent oneshot top10 bacterial cells
    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 <t>Top10</t> 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.
    Competent Oneshot Top10 Bacterial Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher top10 bacteria
    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 <t>Top10</t> 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.
    Top10 Bacteria, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher top10 oneshot cells
    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 <t>Top10</t> 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.
    Top10 Oneshot Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher top10 e
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
    Top10 E, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher escherichiacoli strain top10
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
    Escherichiacoli Strain Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher top10 transformation
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
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    Thermo Fisher coli k 12 derivate top10
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
    Coli K 12 Derivate Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher eschericia coli strain top10
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
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    Thermo Fisher strain top10 electrocomp
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
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    Thermo Fisher top10 vector
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
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    Thermo Fisher ec top10
    Programmed emergence of an RFP-expressing domain across bacterial populations. <t>TOP10</t> E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t
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    Image Search Results


    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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkn884

    Figure Lengend 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).

    Article Snippet: An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression.

    Techniques: Transformation Assay, Plasmid Preparation, Isolation, Agarose Gel 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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkn884

    Figure Lengend 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).

    Article Snippet: An E. coli stain TOP10 (Invitrogen, Carlsbad, CA, USA) ( ) was used as a host for cloning and methyltransferase expression.

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

    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 OD 600 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.

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

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

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

    Figure Lengend 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 OD 600 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.

    Article Snippet: We then characterized the resulting library by transforming the recovered plasmid population into Top10 cells (Invitrogen). shows the diversity of fluorescence intensities obtained, both for individual colonies on an LB agar plate ( panel a ) and for individual cells in suspension ( panel b ).

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

    HPLC separation of mutanolysin-digested peptidoglycan fragments present in the germination exudate of B. megaterium sleB cwlJ (GC103) spores complemented with plasmid-borne SleB variants. Reduced muropeptides were monitored at A 202 and subsequently desalted

    Journal: Journal of Bacteriology

    Article Title: Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ QM B1551 Cortex-Lytic Enzymes ▿ †

    doi: 10.1128/JB.00830-10

    Figure Lengend Snippet: HPLC separation of mutanolysin-digested peptidoglycan fragments present in the germination exudate of B. megaterium sleB cwlJ (GC103) spores complemented with plasmid-borne SleB variants. Reduced muropeptides were monitored at A 202 and subsequently desalted

    Article Snippet: Escherichia coli strains used for site-directed mutagenesis (SDM) (XL1-Blue [Stratagene]) or preparation of plasmids for transformation of B. megaterium (Top10 [Invitrogen]) were cultured at 37°C in LB medium supplemented with 50 μg/ml carbenicillin.

    Techniques: High Performance Liquid Chromatography, Plasmid Preparation

    Germination of B. megaterium GC103 ( sleB cwlJ ) spores complemented with plasmid-borne SleB variants. Spores were heat activated and germinated in buffer (5 mM Tris-HCl, pH 7.8) plus 10 mM glucose, and the OD 600 was recorded. GC103 ( sleB cwlJ ), ⧫;

    Journal: Journal of Bacteriology

    Article Title: Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ QM B1551 Cortex-Lytic Enzymes ▿ †

    doi: 10.1128/JB.00830-10

    Figure Lengend Snippet: Germination of B. megaterium GC103 ( sleB cwlJ ) spores complemented with plasmid-borne SleB variants. Spores were heat activated and germinated in buffer (5 mM Tris-HCl, pH 7.8) plus 10 mM glucose, and the OD 600 was recorded. GC103 ( sleB cwlJ ), ⧫;

    Article Snippet: Escherichia coli strains used for site-directed mutagenesis (SDM) (XL1-Blue [Stratagene]) or preparation of plasmids for transformation of B. megaterium (Top10 [Invitrogen]) were cultured at 37°C in LB medium supplemented with 50 μg/ml carbenicillin.

    Techniques: Plasmid Preparation

    HPLC separation of B. megaterium QM B1551 spore muropeptides. Germination-associated samples were collected 40 min after initiation of germination. Except where indicated, samples were digested with mutanolysin and reduced with sodium borohydride prior

    Journal: Journal of Bacteriology

    Article Title: Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ QM B1551 Cortex-Lytic Enzymes ▿ †

    doi: 10.1128/JB.00830-10

    Figure Lengend Snippet: HPLC separation of B. megaterium QM B1551 spore muropeptides. Germination-associated samples were collected 40 min after initiation of germination. Except where indicated, samples were digested with mutanolysin and reduced with sodium borohydride prior

    Article Snippet: Escherichia coli strains used for site-directed mutagenesis (SDM) (XL1-Blue [Stratagene]) or preparation of plasmids for transformation of B. megaterium (Top10 [Invitrogen]) were cultured at 37°C in LB medium supplemented with 50 μg/ml carbenicillin.

    Techniques: High Performance Liquid Chromatography

    ClustalW alignment of SleB from various Bacillus species. Residues predicted to comprise putative structural domains are denoted. Stars indicate charged residues that were subjected to amino acid substitution in this work. BM, B. megaterium QM B1551;

    Journal: Journal of Bacteriology

    Article Title: Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ QM B1551 Cortex-Lytic Enzymes ▿ †

    doi: 10.1128/JB.00830-10

    Figure Lengend Snippet: ClustalW alignment of SleB from various Bacillus species. Residues predicted to comprise putative structural domains are denoted. Stars indicate charged residues that were subjected to amino acid substitution in this work. BM, B. megaterium QM B1551;

    Article Snippet: Escherichia coli strains used for site-directed mutagenesis (SDM) (XL1-Blue [Stratagene]) or preparation of plasmids for transformation of B. megaterium (Top10 [Invitrogen]) were cultured at 37°C in LB medium supplemented with 50 μg/ml carbenicillin.

    Techniques:

    Muropeptide analysis of dormant and germinating B. megaterium QM B1551 spores.

    Journal: Journal of Bacteriology

    Article Title: Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ QM B1551 Cortex-Lytic Enzymes ▿ †

    doi: 10.1128/JB.00830-10

    Figure Lengend Snippet: Muropeptide analysis of dormant and germinating B. megaterium QM B1551 spores.

    Article Snippet: Escherichia coli strains used for site-directed mutagenesis (SDM) (XL1-Blue [Stratagene]) or preparation of plasmids for transformation of B. megaterium (Top10 [Invitrogen]) were cultured at 37°C in LB medium supplemented with 50 μg/ml carbenicillin.

    Techniques:

    HPLC separation of mutanolysin-digested B. megaterium germination exudates. Peaks are designated as in Table ; “X” denotes primarily nonpeptidoglycan material or buffer components. (a) B. megaterium QM B1551 germination

    Journal: Journal of Bacteriology

    Article Title: Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes ▿ QM B1551 Cortex-Lytic Enzymes ▿ †

    doi: 10.1128/JB.00830-10

    Figure Lengend Snippet: HPLC separation of mutanolysin-digested B. megaterium germination exudates. Peaks are designated as in Table ; “X” denotes primarily nonpeptidoglycan material or buffer components. (a) B. megaterium QM B1551 germination

    Article Snippet: Escherichia coli strains used for site-directed mutagenesis (SDM) (XL1-Blue [Stratagene]) or preparation of plasmids for transformation of B. megaterium (Top10 [Invitrogen]) were cultured at 37°C in LB medium supplemented with 50 μg/ml carbenicillin.

    Techniques: High Performance Liquid Chromatography

    Measured RPU values for promoters assembled in the same plasmid in TOP10 and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).

    Journal: PLoS ONE

    Article Title: Bottom-Up Engineering of Biological Systems through Standard Bricks: A Modularity Study on Basic Parts and Devices

    doi: 10.1371/journal.pone.0039407

    Figure Lengend Snippet: Measured RPU values for promoters assembled in the same plasmid in TOP10 and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).

    Article Snippet: Chemically competent TOP10 (Invitrogen) were cultivated in LB medium and were used as hosts for plasmid propagation, except for pSB4C5(BBa_I52002) which was propagated in chemically competent DB3.1 (Invitrogen), a ccdB toxin-tolerant strain.

    Techniques: Plasmid Preparation, Standard Deviation, Activity Assay, Clone Assay

    Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into TOP10 E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.

    Journal: Molecular microbiology

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol

    doi: 10.1111/j.1365-2958.2006.05216.x

    Figure Lengend Snippet: Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into TOP10 E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.

    Article Snippet: This construct was transformed into TOP10 competent cells (Invitrogen) for expression.

    Techniques: Expressing, Purification, Recombinant, Clone Assay, Plasmid Preparation, Transformation Assay, Affinity Chromatography, SDS Page, Staining, Affinity Column, Activity Assay, Concentration Assay

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0137652

    Figure Lengend 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.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0137652

    Figure Lengend 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.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0137652

    Figure Lengend 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.

    Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).

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

    Programmed emergence of an RFP-expressing domain across bacterial populations. TOP10 E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t

    Journal: Nature Communications

    Article Title: Programmed hierarchical patterning of bacterial populations

    doi: 10.1038/s41467-018-03069-3

    Figure Lengend Snippet: Programmed emergence of an RFP-expressing domain across bacterial populations. TOP10 E . coli were co-transformed by the RFP reporter plasmid p4g3R, a bicistronic controller plasmid pCRB DRT7VTPLux*(500/250/50), and a sender plasmid pSB1C3 I0500 (LuxI/LasI) encoding either luxI or lasI under control of the arabinose-inducible P BAD/araC promoter. Adjacent populations of the different triple-transformed cell types were incubated on membranes placed on minimal nutrient agar which has or has not been supplemented with 25 mM arabinose. Fluorescence intensity of each quadrant at t = 3000 min (time relative to start of incubation), corrected for background signal present in the absence of arabinose, is plotted against position (genotype boundary between quadrants 12 and 13). Error bars represent the s.d. of average values between the 12 equidistant quadrants on each membrane. Corresponding images captured at t

    Article Snippet: All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA).

    Techniques: Expressing, Transformation Assay, Plasmid Preparation, Incubation, Fluorescence

    Ratiometric characterization of T7RNAP activity. a Comparison of different ratiometric reporter plasmids of T7RNAP activity. Ratiometric reporter plasmids combining different variants of the T7 promoter, 5′-UTRs, and origins of replication (underlined) were introduced into T7 Express E . coli for induction curves) was quantified using a plate fluorometer-based assay (see Methods for details), and is reported relative to the absence of inducer. Error bars represent the standard deviation (s.d.) of average values yielded between three biological replicate experiments performed on different days. b Ratiometric characterization of intact and split variants of T7RNAP. TOP10 E . coli were co-transformed by the highest dynamic range ratiometric reporter p4g3VT tested under a and different controller plasmids encoding intact or fragmented variants of the T7RNAP gene (underlined) under control of the arabinose-inducible P BAD/araC for induction curves), and is reported relative to the absence of inducer. Error bars represent the s.d. of average values yielded between three biological replicate experiments performed on different days

    Journal: Nature Communications

    Article Title: Programmed hierarchical patterning of bacterial populations

    doi: 10.1038/s41467-018-03069-3

    Figure Lengend Snippet: Ratiometric characterization of T7RNAP activity. a Comparison of different ratiometric reporter plasmids of T7RNAP activity. Ratiometric reporter plasmids combining different variants of the T7 promoter, 5′-UTRs, and origins of replication (underlined) were introduced into T7 Express E . coli for induction curves) was quantified using a plate fluorometer-based assay (see Methods for details), and is reported relative to the absence of inducer. Error bars represent the standard deviation (s.d.) of average values yielded between three biological replicate experiments performed on different days. b Ratiometric characterization of intact and split variants of T7RNAP. TOP10 E . coli were co-transformed by the highest dynamic range ratiometric reporter p4g3VT tested under a and different controller plasmids encoding intact or fragmented variants of the T7RNAP gene (underlined) under control of the arabinose-inducible P BAD/araC for induction curves), and is reported relative to the absence of inducer. Error bars represent the s.d. of average values yielded between three biological replicate experiments performed on different days

    Article Snippet: All cloning was performed in TOP10 E . coli (Invitrogen, Waltham, MA).

    Techniques: Activity Assay, Standard Deviation, Transformation Assay