Fluorescent Labeling of NanoLuc
Primary Faculty Mentor’s Name
Angie Spencer
Proposal Track
Student
Session Format
Paper Presentation
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 is catalyzed by a class of enzymes known as luciferases. Luciferases are utilized in a wide variety of molecular biology applications including Bioluminescence Resonance Energy Transfer (BRET). BRET is a technique based 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 enough proximity (approximately 10 nm) and their emission and absorbance spectra overlap, the acceptor molecule absorbs the energy from the donor molecule resulting in the emission of light at a longer wavelength. This spectral shift can be measured and quantified, allowing the study of molecular events such as protein-protein interactions. 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. In order to improve upon current BRET techniques, purified Nluc is being tagged with various fluorescent dyes to determine which of these molecules can act as the best energy acceptor in BRET with Nluc as the energy donor.
Keywords
NanoLuc, fluorescence, bioluminescence
Location
Room 1909
Presentation Year
2015
Start Date
11-7-2015 9:00 AM
End Date
11-7-2015 10:00 AM
Publication Type and Release Option
Presentation (Open Access)
Recommended Citation
DuPlain, Holly, "Fluorescent Labeling of NanoLuc" (2015). Georgia Undergraduate Research Conference (2014-2015). 39.
https://digitalcommons.georgiasouthern.edu/gurc/2015/2015/39
Fluorescent Labeling of NanoLuc
Room 1909
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 is catalyzed by a class of enzymes known as luciferases. Luciferases are utilized in a wide variety of molecular biology applications including Bioluminescence Resonance Energy Transfer (BRET). BRET is a technique based 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 enough proximity (approximately 10 nm) and their emission and absorbance spectra overlap, the acceptor molecule absorbs the energy from the donor molecule resulting in the emission of light at a longer wavelength. This spectral shift can be measured and quantified, allowing the study of molecular events such as protein-protein interactions. 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. In order to improve upon current BRET techniques, purified Nluc is being tagged with various fluorescent dyes to determine which of these molecules can act as the best energy acceptor in BRET with Nluc as the energy donor.
