• Assessment of BRET between NanoLuc and Various Fluorescent Dyes

      DuPlain, Holly; Department of Chemistry and Physics (2016-03)
      Bioluminescence is a natural phenomenon whereby light is emitted by a living organism. This light is generated when a sub- strate is reacted upon by enzymes called luciferases. Bioluminescence Resonance Energy Transfer (BRET) is a technique that relies on a luciferase (donor) to transfer energy to a fluorescent molecule (acceptor). If the donor and acceptor are in close proximity and their emission and excitation spectra overlap, the acceptor absorbs energy from the donor and light is emitted at a longer wavelength. This spectral shift can be quantified. One such luciferase is NanoLuc (Nluc), a genetically engineered en- zyme from Oplophorus gracilirostris. In order to explore the use of Nluc as a donor in BRET, we cloned the gene for Nluc into the plasmid vector pET21c(+). Formation of recombinant DNA was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, Nluc protein containing a C-terminal His6 tag was over-expressed and purified using affinity chromatography. Purification yielded a relatively pure protein with a molecular weight of 19 kDa as judged by SDS-PAGE. Activity of the protein was verified by measuring its ability to generate light in the presence of coelenterazine. The ability of Nluc in conjunction with various acceptors, both attached and free at varying concentrations, to perform BRET will be assessed using luminometry and fluorescence spectroscopy. Funding Source: Center for Undergraduate Research Summer Scholars Program and Department of Chemistry and Physics