Fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments

    Publikation: Konferencebidrag uden forlag/tidsskriftPosterForskningpeer review

    Abstract

    Background: The study of horizontal gene transfer (HGT) in microbial communities has been revolutionized by advances in cultivation-independent methods based on fluorescence reporter-gene technologies. However, the use of fluorescent markers like green fluorescent protein (GFP) and mCherry is limited by environmental constraints that affect the correct maturation of their fluorophores, such as oxygen availability and pH levels. Few studies have focused on elucidating their impact, and the sheer amount of distinct protein variants requires each system to be examined in an individual fashion. The wealth of ecologically and clinically relevant oxygen-deprived micro-habitats in which bacteria thrive, calls for the urgent development of suitable tools that permit their study.

    Objectives: Development of an aerobic fluorescence recovery method for mCherry and GFPmut3, as well as characterisation of the impact the pH has on their fluorescence intensities. Validation of the dual-labelling system for the study of HGT in anoxic milieus.

    Methods: The time-course fluorescent recovery of mCherry and GFPmut3 in vivo, as well as the effect of the extracellular pH on fluorescence was monitored through flow cytometry. The applicability of the findings was validated in anaerobic filter mating experiments.

    Conclusions: The findings present a solution to an intrinsic problem that has long hampered the utilization of this system to study HGT in environments devoid of oxygen, highlight its pH limitations, and provide the experimental tools that will help broaden its horizon of application to other fields.


    OriginalsprogEngelsk
    Publikationsdato1 jun. 2017
    StatusUdgivet - 1 jun. 2017
    BegivenhedFEMS 2017: 7th Congress of European Microbiologists - Spain, Valencia, Spanien
    Varighed: 9 jul. 201713 jul. 2017
    http://www.fems-microbiology2017.kenes.com/

    Konference

    KonferenceFEMS 2017
    LokationSpain
    LandSpanien
    ByValencia
    Periode09/07/1713/07/17
    Internetadresse

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