A Strategy to Purify and Express the Restriction Endonuclease BcnI

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Restriction endonucleases, also known as restriction enzymes, are an essential class of enzymes that cleave the phosphodiester bond of polynucleotide chains at or near a specific nucleotide sequence. Due to the variety of mechanisms endonucleases can enact, they have been classified into four types. Type II restriction endonucleases are typically used in lab experiments because they cut DNA at defined positions producing distinct fragments and gel banding patterns. The mechanism in which these enzymes interact with DNA, specifically the differences between monomers and dimers, are unknown. Furthermore, there has not been a universal protocol made to produce different varieties of Type II restriction endonucleases so that DNA-protein interactions can be observed. We have used Infusion Cloning to insert a synthetic gene encoding the restriction endonuclease for BcnI into a plasmid and generated an expression vector for this protein of interest. Expression of the synthetic BcnI gene from the expression vector is under the control of the LacI operon and T7 promoter. Protein expression will be produced by transforming the BcnI expression vector into E.coli. This expression will be regulated through the LacI operon using glucose as well as the lactose analog IPTG to turn expression on and off. If this strategy is successful, it will yield a universal protocol for protein expression that will permit any restriction endonuclease to be studied. Here we demonstrate successful development of a BcnI expression vector construct as well as the first steps in an induction and purification protocol. This research is an essential step in a larger project that aims to quantify and describe DNA-endonuclease interactions using Förster Resonance Energy Transfer (FRET), which quantifies the energy transfer between two components, and Total Internal Reflection Fluorescence (TIRF) microscopy.

    Item Description
    Name(s)
    Date
    April 15, 2017
    Extent
    57 pages
    Language
    eng
    Genre
    Physical Form
    electronic
    Discipline
    Rights and Use
    In Copyright – Non-Commercial Use Permitted
    Restrictions on Use
    Access restricted until April 15, 2023. Please contact wesscholar@wesleyan.edu for more information.
    Digital Collection
    PID
    ir:1873