• Login
    View Item 
    •   DSpace@RPI Home
    • Rensselaer Libraries
    • z_Technical Services
    • View Item
    •   DSpace@RPI Home
    • Rensselaer Libraries
    • z_Technical Services
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Dynamic regulation of metabolic pathways with crispr-dcas9 toehold-gated rna regulators

    Author
    Perl, Alexander, J
    View/Open
    Perl_rpi_0185E_12155.pdf (2.088Mb)
    Other Contributors
    Koffas, Mattheos; Bystroff, Christopher; Royer, Catherine; Bonocora, Richard;
    Date Issued
    2022-12
    Subject
    Biochemistry and biophysics
    Degree
    PhD;
    Terms of Use
    This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute (RPI), Troy, NY. Copyright of original work retained by author.;
    Metadata
    Show full item record
    URI
    https://hdl.handle.net/20.500.13015/6358
    Abstract
    In metabolic engineering, once a metabolic pathway is expressed, there is an optimization process that aims to increase the production, titer, rate, and yield of the target molecule with a goal to direct the maximum possible amount of metabolic flux toward the pathway of interest. Our labs were able to take the dCas9 system and incorporate the spacer into a modified toehold switch creating a toehold-gated sgRNA (thgRNA) which acts as an orthogonal, transcriptional regulator when activated by an endogenous trigger strand. This system was used to show transcriptional repression of simple and complex metabolic pathways in Escherichia Coli (E. coli). We also looked at a variety of factors including: promoter strength, temperature, and induction time on mCherry fluorescence and violacein production. Previously, we introduced the violacein pathway, a five-step metabolic pathway, into a pETM6 vector with weakened T7 promoter. By incorporating spacers targeting these weakened promoters into thgRNA, we were able to show repression of individual and multiple genes. Our thgRNA are able to function as a conditional activation of CRISPR-based systems by using highly predictable toehold-mediated strand displacement reactions. Sequence specific unblocking of the spacer allows for both orthogonality and low cross-talk between thgRNA. Additionally, these devices do not require the screening of large libraries currently needed to create such specific riboregulators as the CRISPR spacers are so sequence specific.;
    Description
    December 2022; School of Science
    Department
    Biochemistry and Biophysics Program;
    Publisher
    Rensselaer Polytechnic Institute, Troy, NY
    Relationships
    Rensselaer Theses and Dissertations Online Collection;
    Access
    Restricted to current Rensselaer faculty, staff and students in accordance with the Rensselaer Standard license. Access inquiries may be directed to the Rensselaer Libraries.;
    Collections
    • z_Technical Services

    Browse

    All of DSpace@RPICommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    Login

    DSpace software copyright © 2002-2023  DuraSpace
    Contact Us | Send Feedback
    DSpace Express is a service operated by 
    Atmire NV