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    Engineering a split-GFP system for direct pathogen detection

    Author
    Banerjee, Shounak
    View/Open
    177446_Banerjee_rpi_0185E_10950.pdf (5.742Mb)
    Other Contributors
    Bystroff, Christopher, 1960-; Garde, Shekhar; Wang, Chunyu; Barquera, Blanca L.;
    Date Issued
    2016-08
    Subject
    Biology
    Degree
    PhD;
    Terms of Use
    This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.;
    Metadata
    Show full item record
    URI
    https://hdl.handle.net/20.500.13015/1740
    Abstract
    The process often requires mutations of residues surrounding the chromophore and could lead to a loss of function and destabilization. Therefore, four different studies were initiated and are described. The first set of studies was to test the effects of restoring an internal hydrogen bonding network, disulfide engineering and introducing insertions and deletions in GFP. The second study was to test the tolerance of the protein’s chromophore’s micro-environment to point mutations. The saturation mutagenesis studies thus performed, revealed that the microenvironment is surprisingly robust. The third study, was to develop several biosensor prototypes. Two of these produced promising results and are described in detail. A good LOO-GFP biosensor should ideally have no fluorescence in the absence of the target but the prototypes developed thus far show high levels of background fluorescence due to oligomerization of unbound LOO-GFP. One way to mitigate this is to use a genetic fusion based immobilization approach to tether LOO-GFPs to a self-assembled protein matrix. This was the subject of the fourth study. Preliminary results showed that the genetic fusion did not compromise fluorescent functionality in the LOO-GFP. Thus in summary, the results of the four studies outlined above, produced results showing good promise for the engineering of LOO-GFPs to directly interact with protein targets of interest.; The green fluorescent protein (GFP) has seen widespread use in biological research. It can be split into two or three fragments, which self-assemble and reconstitute structure and function. A particular type of split GFP is called Leave-one-out-GFP (LOO-GFP). A LOO-GFP is designed to reconstitute fluorescent function upon exogenous addition of its left-out piece. In this work, efforts toward modifying LOO-GFPs to reconstitute function upon binding specifically to a protein of interest are described. This could enable the use of LOO-GFPs as versatile biosensors for direct pathogen detection. Computational protein design algorithms were used to calculate the set of mutations required to enable the LOO-GFP-target interaction.;
    Description
    August 2016; School of Science
    Department
    Dept. of Biological Sciences;
    Publisher
    Rensselaer Polytechnic Institute, Troy, NY
    Relationships
    Rensselaer Theses and Dissertations Online Collection;
    Access
    Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.;
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    • RPI Theses Online (Complete)

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