Hdl Handle:
http://hdl.handle.net/10675.2/601080
Title:
Assessment of BRET between NanoLuc and Various Fluorescent Dyes
Authors:
DuPlain, Holly
Abstract:
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
Affiliation:
Department of Chemistry and Physics
Issue Date:
Mar-2016
URI:
http://hdl.handle.net/10675.2/601080
Type:
Presentation
Language:
en_US
Description:
Presentation given at the 17th Annual Phi Kappa Phi Student Research and Fine Arts Conference
Appears in Collections:
17th Annual Phi Kappa Phi Student Research and Fine Arts Conference: Oral Symposia II

Full metadata record

DC FieldValue Language
dc.contributor.authorDuPlain, Hollyen
dc.date.accessioned2016-03-09T21:05:46Zen
dc.date.available2016-03-09T21:05:46Zen
dc.date.issued2016-03en
dc.identifier.urihttp://hdl.handle.net/10675.2/601080en
dc.descriptionPresentation given at the 17th Annual Phi Kappa Phi Student Research and Fine Arts Conferenceen
dc.description.abstractBioluminescence 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 Physicsen
dc.language.isoen_USen
dc.subjectBioluminescence Resonance Energy Transfer Techniquesen
dc.subjectLuciferasesen
dc.subjectSpectrometry, Fluorescenceen
dc.titleAssessment of BRET between NanoLuc and Various Fluorescent Dyesen_US
dc.typePresentationen
dc.contributor.departmentDepartment of Chemistry and Physicsen
dc.description.advisorSpencer,Angela; Lambert, Nevinen
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