Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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 Engineering of Geranyl Diphospate C-Methyltransferase for the Development of New Diterpenoid Precursors(Izmir Institute of Technology, 2014) Akıl, Caner; Köksal, Mustafa; Köksal, MustafaTerpenoids constitute the most diverse family of natural products. They are involved in several biological functions and are used in medical and industrial applications. The key to their diverse biological activities is their structural diversity. Terpenoids are synthesized in three stages, all of which contribute to generation of structural diversity. In the terpenoid biosynthetic pathways, terpene synthases generate larger linear terpenoid precursors from smaller units via condensation reactions, terpene cyclases transform precursors via cyclization reactions, and then tailoring enzymes modify terpenoid products via addition of functional groups. Recently discovered geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is able to modify a linear monoterpenoid precursor, geranyl diphoshate (GPP), to produce a non-canonical terpenoid precursor, 2-methylgeranyl diphosphate. Modification of GPP by GPPMT is the first example of modification of a canonical linear isoprenoid precursor in nature. This study aims to achieve enzymatic synthesis of novel methylated non-canonical diterpenoid precursors, such as 2-methylgeranylgeranyl diphosphate (2MGGPP) by engineering GPPMT. The novel non-canonical precursors may later be utilized by cyclases to enhance the diversity of the terpenome. For example, taxadiene synthase could utilize 2MGGPP to generate variants of taxadiene, the precursor of the leading anti-cancer drug paclitaxel (Taxol®). Candidate mutants predicted to use GGPP as substrate were selected via in silico analysis of GPPMT structure. These mutations were introduced using the Quick-change site-directed mutagenesis. Mutant genes were expressed in E.coli strains. Mutant proteins were purified by Fast Protein Liquid Chromatography. Catalytic activities of mutants against canonical terpenoid precursors were determined by SAM methyltransferase assay.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.