Hdl Handle:
http://hdl.handle.net/10675.2/583084
Title:
Cloning, Over-expression and Purification of Nanoluc
Authors:
DuPlain, Holly; Parks, Jasmine; Blocker, Brittany
Abstract:
Bioluminescence is a natural phenomenon that occurs in bacteria, insects, fungi and some marine species whereby a living organism emits light via a chemical reaction catalyzed by enzymes called luciferases. Luciferases are utilized in various applications including Bioluminescence Resonance Energy Transfer (BRET). BRET uses a luciferase (energy donor) to transfer energy to a fluorescent protein or dye (energy acceptor). If the donor and acceptor are close together and their emission and absorbance spectra overlap, the acceptor absorbs the energy from the donor and light is emitted at a longer wavelength. This spectral shift is measured. One such luciferase is Nanoluc (Nluc), a genetically engineered enzyme from the deep sea shrimp Oplophorus gracilirostris. To explore the use of Nluc as a BRET energy donor, the Nluc gene was cloned into the plasmid vector pET21c(+). Recombinant DNA formation was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, C-terminal His6 tagged Nluc protein 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. Protein activity was assessed by measuring its ability to generate light in the presence of the substrate coelenterazine. Begin Time: 08:25 End Time: 31:16
Affiliation:
College of Science and Mathematics
Issue Date:
13-Nov-2015
URI:
http://hdl.handle.net/10675.2/583084
Additional Links:
https://lecture.gru.edu/ess/echo/presentation/c6aaf27c-db4b-4035-a58a-20de0b0f7266?ec=true
Type:
Presentation
Language:
en_US
Description:
Presentation given at the CURS Brown Bag Seminar Series on November 13, 2015
Series/Report no.:
Fall; 2015
Sponsors:
Center for Undergraduate Research and Scholarship; College of Science and Mathematics; Department of Chemistry and Physics
Appears in Collections:
CURS Brown Bag Presentations

Full metadata record

DC FieldValue Language
dc.contributor.authorDuPlain, Hollyen
dc.contributor.authorParks, Jasmineen
dc.contributor.authorBlocker, Brittanyen
dc.date.accessioned2015-12-01T20:00:15Zen
dc.date.available2015-12-01T20:00:15Zen
dc.date.issued2015-11-13en
dc.identifier.urihttp://hdl.handle.net/10675.2/583084en
dc.descriptionPresentation given at the CURS Brown Bag Seminar Series on November 13, 2015en
dc.description.abstractBioluminescence is a natural phenomenon that occurs in bacteria, insects, fungi and some marine species whereby a living organism emits light via a chemical reaction catalyzed by enzymes called luciferases. Luciferases are utilized in various applications including Bioluminescence Resonance Energy Transfer (BRET). BRET uses a luciferase (energy donor) to transfer energy to a fluorescent protein or dye (energy acceptor). If the donor and acceptor are close together and their emission and absorbance spectra overlap, the acceptor absorbs the energy from the donor and light is emitted at a longer wavelength. This spectral shift is measured. One such luciferase is Nanoluc (Nluc), a genetically engineered enzyme from the deep sea shrimp Oplophorus gracilirostris. To explore the use of Nluc as a BRET energy donor, the Nluc gene was cloned into the plasmid vector pET21c(+). Recombinant DNA formation was verified by agarose gel electrophoresis. After transformation of the recombinant plasmid into E. coli BL21 cells, C-terminal His6 tagged Nluc protein 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. Protein activity was assessed by measuring its ability to generate light in the presence of the substrate coelenterazine. Begin Time: 08:25 End Time: 31:16en
dc.description.sponsorshipCenter for Undergraduate Research and Scholarship; College of Science and Mathematics; Department of Chemistry and Physicsen
dc.language.isoen_USen
dc.relation.ispartofseriesFallen
dc.relation.ispartofseries2015en
dc.relation.urlhttps://lecture.gru.edu/ess/echo/presentation/c6aaf27c-db4b-4035-a58a-20de0b0f7266?ec=trueen
dc.subjectBioluminescenceen
dc.subjectLuciferasesen
dc.subjectElectrophoresis, Agar Gelen
dc.subjectDNA, Recombinanten
dc.titleCloning, Over-expression and Purification of Nanolucen_US
dc.typePresentationen
dc.contributor.departmentCollege of Science and Mathematicsen
dc.contributor.mentorSpencer, Angieen
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