Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
Browse
3 results
Search Results
Master Thesis Expression, Purification and Preliminary Functional Characterization of the Acidianus Two-Tailed Virus Tnpb Endonucleases(01. Izmir Institute of Technology, 2024) Burgeia, Arwa Saleh; Oke, MuseTnpB is an RNA-guided endonuclease encoded in the IS200/605 transposon family. It forms a complex with a self-encoded ωRNA that directs TnpB to an appropriate target DNA sequence for activity. DNA cleavage at the target site promotes homologous recombination thus ensuring propagation of IS200/605 elements. Although IS200/605 elements are abundant in prokaryotic genomes, they are rarely found in viruses. However, the Acidianus two-tailed virus (ATV), which possesses 72 genes, encodes four TnpB proteins (gp10, gp40, gp43 and gp68). In this study, bioinformatics analysis of all ATV tnpB genes demonstrated that they were all solo tnpB genes and therefore could be classified as part of the IS1341 group. TnpB proteins are characterized by having a DED catalytic site motif and a C-terminal C4 zinc finger motif. Although the ATV gp10 protein retains the DED active site motif found in most TnpB proteins, the other ATV TnpB proteins possess a different amino acid instead of the Glu residue. Additionally, the ATV TnpB proteins possess the C4 zinc finger motif except for ATV gp40, which possesses three cysteine residues. All four ATV tnpB genes were cloned and expressed in the heterologous Escherichia coli host. The gp40 and gp68 proteins were purified using Ni2+ -NTA and gel filtration chromatography. Although gp68 appears to bind to DNA, there is insufficient evidence for RNA binding. Cleavage assays revealed that gp68 demonstrated nonspecific DNA nickase and cleavage activities. The significance of this study and the broader implications regarding the possible role of TnpB in virus survival are discussed.Master Thesis Cloning, Heterologous Expression and Purification of Various Wax Ester Synthases in Escherichia Coli(Izmir Institute of Technology, 2017) Ovacık, Kamil; Arslanoğlu, AlperBiodiesel, known all around theWorld, is a diesel fuel containing fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs) with different molecular weights. The recent studies which are about the development of FAEE focused on production of FAEEs in vivo syntheses. This synthesis is catalyzed by wax ester synthases (WS). Bifunctional wax ester synthase/acyl-coenzyme-A (acyl-CoA): diacylglycerol acyltransferase (WS/DGAT) synthesizes wax ester by processing a certain range of fatty alcohols and fatty acyl-CoAs. It is considered as the final enzyme in biosynthetic process of wax ester production. Aim of the research is cloning, heterologous expression, purification and crystallization trial of was ester synthases from M. aquaeolei VT8 (MaWES) and R. opacus PD630 (RoWES). MaWES was cloned into pET expression vector and heterologous expression of MaWES was carried out in E.coli BL21 (DE3) strain. Three chromatography systems were used for purification of MaWES. After Immobilized Metal Affinity Chromatography (IMAC), buffer exchange and gel filtration chromatography, enzyme was purified with approximately 100 mg yield. This project can pave the way for structural studies WS/DGAT enzymes mentioned above. In summary, the findings of this study will circuitously help for solving the relationship between function and structure of these enzymes. It may lead to increased generation of FAEE based biodiesel.Master Thesis Synthesis, Characterization of Cdsxse1-X Quantum Dots and Evaluation of Their Real-Time Motions in Live Cells(Izmir Institute of Technology, 2011) Ünal, Gülçin; Özçelik, SerdarThe use of quantum dots as fluorescent labels in bioimaging is the most intensively studied subject. The aim of this study is to elucidate locations of quantum dots and track their motions in real time through confocal microscopy and to evaluate influence of surface chemistry on diffusions of quantum dots in live cells. In this study, trioctylphosphine oxide (TOPO) capped CdSxSe1-x quantum dots were synthesized and then TOPO molecules were exchanged with 3-mercaptopropionic acid and N-acetyl-Lcysteine to make quantum dots water dispersible for cellular imaging. Human lung adenocarcinoma epithelial cells (A549) and human bronchial epithelial cells (BEAS-2B) were incubated 1 hour with CdSxSe1-x quantum dots with a concentration range of 1-10 g/mL. Localizations and real time motions of quantum dots were tracked by a spinning disc confocal microscope. The center of fluorescent spots of quantum dots was determined by 2D Gaussian fitting with a sub-pixel resolution (<100nm/pixel). The mean square displacements, diffusion coefficients and trajectories in which quantum dots made motions were analyzed by the software ImageJ with a plug in Spot Tracker. Confocal images showed that both MPA and NAC cappped quantum dots were observed in the cytoplasm of cells. Trajectories of quantum dots in cellular environment demonstrated that the quantum dots performed various types of motions in live cells. Unimodal, trimodal and multimodal distribution histograms of the diffusion coefficeints were obtained for different capping agents (MPA and NAC) and cell types (A549 and BEAS-2B). We conclude that the surface chemistry regulates the motion of the quantum dots in the cellular environment.