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conventional wide field fluorescence microscopy imaging  (Nikon)


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    Nikon conventional wide field fluorescence microscopy imaging
    Visualization and model of TAP activity. ( A ) <t>Fluorescence</t> <t>microscopy</t> images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.
    Conventional Wide Field Fluorescence Microscopy Imaging, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 10098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Specific killing and resensitization of pathogenic Escherichia coli strains carrying bla CTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs)"

    Article Title: Specific killing and resensitization of pathogenic Escherichia coli strains carrying bla CTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaf1466

    Visualization and model of TAP activity. ( A ) Fluorescence microscopy images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.
    Figure Legend Snippet: Visualization and model of TAP activity. ( A ) Fluorescence microscopy images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.

    Techniques Used: Activity Assay, Fluorescence, Microscopy, Injection, Conjugation Assay, Plasmid Preparation, CRISPR, Expressing



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    Visualization and model of TAP activity. ( A ) <t>Fluorescence</t> <t>microscopy</t> images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.
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    Visualization and model of TAP activity. ( A ) Fluorescence microscopy images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.

    Journal: Nucleic Acids Research

    Article Title: Specific killing and resensitization of pathogenic Escherichia coli strains carrying bla CTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs)

    doi: 10.1093/nar/gkaf1466

    Figure Lengend Snippet: Visualization and model of TAP activity. ( A ) Fluorescence microscopy images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.

    Article Snippet: Ctx was added and cells were imaged every 10 min for 3 h. Conventional wide-field fluorescence microscopy imaging was carried out on an Eclipse Ti2-E microscope (Nikon), equipped with x100/1.45 oil Plan Apo Lambda phase objective, ORCA-Fusion digital CMOS camera (Hamamatsu), and using NIS software for image acquisition.

    Techniques: Activity Assay, Fluorescence, Microscopy, Injection, Conjugation Assay, Plasmid Preparation, CRISPR, Expressing