Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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, Muse; Oke, Muse; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyTnpB 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, Alper; Arslanoğlu, Alper; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBiodiesel, 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, Mustafa; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyTerpenoids 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.