DNA-based assembly of transcription factors for bioluminescence resonance energy transfer (BRET) assay /

Kazan, Hasan Hüseyin
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.


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Oral, Çağatay Mert; Ercan, Batur; Department of Metallurgical and Materials Engineering (2020)
Calcium carbonate (CaCO3) particles have been widely used in biomedical applications owing to their biocompatibility and biodegradability. In order to effectively utilize CaCO3 particles in biomedical applications, their physical and chemical properties should be systematically controlled. However, this is a challenging task due to the presence of three different anhydrous CaCO3 polymorphs having complex crystallization behavior. In this thesis, CaCO3 particles were synthesized at distinct environments to c...
Thermal properties of Deng-Fan-Eckart potential model using Poisson summation approach
Edet, C. O.; Okorie, U. S.; Osobonye, G.; Ikot, A. N.; Rampho, G. J.; Sever, Ramazan (Springer Science and Business Media LLC, 2020-05-01)
The Deng-Fan-Eckart potential is as good as the Morse potential in studying atomic interaction in diatomic molecules. By using the improved Pekeris-type approximation, to deal with the centrifugal term, we obtain the bound-state solutions of the radial Schrodinger equation with this adopted molecular model via the Factorization Method. With the energy equation obtained, the thermodynamic properties of some selected diatomic molecules (H-2, CO, ScN and ScF) were obtained using Poisson summation method. The u...
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Chakraborti, N.; Das, S.; Jayakanth, R.; Pekoz, R.; Erkoç, Şakir (Informa UK Limited, 2007-01-01)
Empirical potentials based upon two and three body interactions were applied to the Li+ -C system, assuming the Li+ ions to be distributed inside high-symmetry, single walled carbon nanotubes of different chirality. Structural optimizations for various assemblages were conducted using evolutionary and genetic algorithms, where differential evolution and particle swarm optimization techniques worked satisfactorily. The results were compared with the outcome of some rigorous molecular dynamics simulations. Th...
Experimental Realization of Single Current Diffusion Tensor Magnetic Resonance Electrical Impedance Tomography
Sadighi, Mehdi; Şişman, Mert; Açıkgöz, Berk Can; Eyüboğlu, Behçet Murat (2020-08-08)
To obtain low-frequency anisotropic conductivity distribution of biological tissues recently Diffusion Tensor Magnetic Resonance Electrical Impedance Tomography (DT-MREIT), which is combination of DTI and MREIT techniques, is proposed. There are two in vivo applications of DT-MREIT in the literature where two linearly independent current injection patterns are used. Decreasing the number of current injection patterns to one improves the feasibility of DT-MREIT in clinical applications. In this study, DT-MRE...
Optical absorption of a quantum well with an adjustable asymmetry
Yildirim, H.; Tomak, Mehmet (Springer Science and Business Media LLC, 2006-04-01)
The effects of asymmetry and the electric field on the electronic subbands and the nonlinear intersubband optical absorption of GaAs quantum wells represented by a Poschl-Teller confining potential are studied. The potential itself can be made asymmetric through a correct choice of its parameter set and this adjustable asymmetry is important for optimizing the absorption. In that way optimal cases can be created. We calculate the modified wave functions and electronic subbands variationally. The linear and ...
Citation Formats
H. H. Kazan, “DNA-based assembly of transcription factors for bioluminescence resonance energy transfer (BRET) assay /,” M.S. - Master of Science, Middle East Technical University, 2015.