Date

2015

Author

Kazan, Hasan Hüseyin

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Bioluminescence Resonance Energy Transfer (BRET) is a promising assay for studying molecular dynamics such as protein-protein interactions especially in situ and in vivo since the system requires precise distance between the molecules. BRET technique has been used for identification of molecular interactions in situ, imaging of deep-tissues in animal models and sensing of organic or inorganic molecules in vitro by combining the luciferase derivative that is an obligatory unit for this assay and any fluorescent molecules, such as inorganic fluorescent molecules and fluorescent proteins. Beside the luciferase derivatives must be used as one part of BRET pairs, the overall system would be optimized to adapt desired properties by changing the substrate of the luciferase and fluorescent molecule with suitable excitation and emission wavelength as well as the platform that locates the BRET pairs particularly for sensing studies via BRET technique. As sharing the perspective of Fluorescence Resonance Energy Transfer (FRET) which was described previously and has been used widely in molecular interactions, BRET systems have been established basically on in situ studies; however, BRET could be used in sensing systems more widely thanks to its advantages over FRET technique. Deoxyribonucleic acid (DNA) has been shown as the source of life; nonetheless, it is a remarkable bio-polymer having stable and predictable structure with cheap production of synthetic variants. Basing on these useful properties of DNA, it has been used popularly to develop scaffold to combine molecules with exact distances for any purposes or proof of concept studies using molecular self-assembly principle. In the present study, we aimed to design a BRET assay in which engineered transcription factors are fused to BRET pairs, Renilla luciferase (RLuc) and fluorescent protein, mCherry, and signaled on DNA scaffold as a proof of concept study. We fused complementary DNA (cDNA) of CDC DNA-binding protein that is the engineered form of human estrogen receptor α (hERα) to cDNA of RLuc and cDNA of yeast protein, Gal4 DNA-binding domain to cDNA of mCherry with the common protein purification tag of 6-Histidine (6xHis) in a bacterial expression vector by cloning studies. Upon construction of the plasmids that code for related fusion proteins, we tried to over-express, isolate and purify the fusion proteins for next steps of the study and the final process was confirmed by Western Blot (WB) analysis. As an ongoing study, we will try to verify the binding of proteins to DNA scaffold by biochemical methods. Next, we will try to obtain BRET signal and optimize the overall system. This platform would be adapted to in situ and regarded to be used to study context-dependent transcription machinery, genome editing and protein binding to DNA.

Subject Keywords

DNA., Bioluminescence., Bioluminescence assay.

URI

http://etd.lib.metu.edu.tr/upload/12618422/index.pdf
https://hdl.handle.net/11511/24387

Collections

Graduate School of Natural and Applied Sciences, Thesis