Hdl Handle:
http://hdl.handle.net/10675.2/565704
Title:
Cloning, Expression, and Purification of Nanoluc
Authors:
DuPlain, Holly; Parks, Jasmine; Blocker, Brittany; Spencer, Angie
Abstract:
Bioluminescence is a natural phenomenon that occurs in bacteria, insects, fungi and some marine species whereby light is emitted by a living organism. This emitted light is generated by a chemical reaction that occurs when a substrate is reacted upon by a class of enzymes called luciferases. Bioluminescence Resonance Energy Transfer (BRET) is a technique that relies on the use of a luciferase (energy donor) to transfer energy to a nearby fluorescent protein or dye (energy acceptor). If the donor and acceptor molecules are in close proximity and their emission and absorbance spectra overlap, the acceptor absorbs the energy from the donor (luciferase) which results in the emission of light at a longer wavelength (i.e. different color). This spectral shift can be measured and quantified. Because of the widespread applications and utility of luciferase enzymes, many assay systems have been developed that make use of various luciferases as energy donors. One such luciferase is Nanoluc (Nluc), a genetically engineered enzyme from the deep sea shrimp, Oplophorus gracilirostris. In order to explore the use of Nluc as an energy donor in BRET, we cloned the gene for Nluc into the plasmid vector, pET21c(+). The formation of the recombinant DNA was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, the Nluc protein containing a C-terminal His6 tag was over-expressed and purified using affinity chromatography. The purification yielded a relatively pure protein with a molecular weight of 19 kDa as judged by SDS-PAGE. The activity of the protein was assessed by measuring its ability to generate light in the presence of the substrate coelenterazine.
Affiliation:
College of Science and Mathematics
Issue Date:
7-Aug-2015
URI:
http://hdl.handle.net/10675.2/565704
Type:
Presentation
Description:
Poster presentation given at the 2015 CURS Summer Scholars Symposium
Sponsors:
Office of the Provost, VP for Academic and Faculty Affairs, Office of Research
Appears in Collections:
Summer Scholars Program

Full metadata record

DC FieldValue Language
dc.contributor.authorDuPlain, Hollyen
dc.contributor.authorParks, Jasmineen
dc.contributor.authorBlocker, Brittanyen
dc.contributor.authorSpencer, Angieen
dc.date.accessioned2015-08-07T00:12:39Zen
dc.date.available2015-08-07T00:12:39Zen
dc.date.issued2015-08-07en
dc.identifier.urihttp://hdl.handle.net/10675.2/565704en
dc.descriptionPoster presentation given at the 2015 CURS Summer Scholars Symposiumen
dc.description.abstractBioluminescence is a natural phenomenon that occurs in bacteria, insects, fungi and some marine species whereby light is emitted by a living organism. This emitted light is generated by a chemical reaction that occurs when a substrate is reacted upon by a class of enzymes called luciferases. Bioluminescence Resonance Energy Transfer (BRET) is a technique that relies on the use of a luciferase (energy donor) to transfer energy to a nearby fluorescent protein or dye (energy acceptor). If the donor and acceptor molecules are in close proximity and their emission and absorbance spectra overlap, the acceptor absorbs the energy from the donor (luciferase) which results in the emission of light at a longer wavelength (i.e. different color). This spectral shift can be measured and quantified. Because of the widespread applications and utility of luciferase enzymes, many assay systems have been developed that make use of various luciferases as energy donors. One such luciferase is Nanoluc (Nluc), a genetically engineered enzyme from the deep sea shrimp, Oplophorus gracilirostris. In order to explore the use of Nluc as an energy donor in BRET, we cloned the gene for Nluc into the plasmid vector, pET21c(+). The formation of the recombinant DNA was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, the Nluc protein containing a C-terminal His6 tag was over-expressed and purified using affinity chromatography. The purification yielded a relatively pure protein with a molecular weight of 19 kDa as judged by SDS-PAGE. The activity of the protein was assessed by measuring its ability to generate light in the presence of the substrate coelenterazine.en
dc.description.sponsorshipOffice of the Provost, VP for Academic and Faculty Affairs, Office of Researchen
dc.subjectNanolucen
dc.titleCloning, Expression, and Purification of Nanolucen
dc.typePresentationen
dc.contributor.departmentCollege of Science and Mathematicsen
All Items in Scholarly Commons are protected by copyright, with all rights reserved, unless otherwise indicated.