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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Měsíčková, Klára ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
The isolation of plasmid DNA is an important and often used method in microbiology. The isolation itself is preceded by preparation of bacterial competent cells and by amplification of the plasmids. In this stage, plasmids CHR2, ASAP1, ASAP-3, ASAP-5 and Kir2.1. are first amplified in E.Coli bacteria of the DH5 strain and then isolated through the method of phenol-chloroform extraction. Gel electrophoresis and transfection to cellular line HEK293 are used for determining the correctness of the isolation.
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Preparation and purification of plasmid DNAs
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of therapeutic methods in medicine, like are DNA vaccines and gene therapy increases demand for new processes for the isolation and purification highly pure plasmid DNA. Most often used methods of purification plasmid DNA are chromatographic methods. In experimental part of this thesis was performed isolation of plasmid pUC-19 DNA plasmid via alkalyne lysis. And purification of plasmid was performed by liquid chromatography and agarose gel electrophoresis.
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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Bačovská, Kristýna ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
Plasmid DNA is commonly used in fields of molecular biology and genetic engineering. This work deals with methods of DNA isolation and topics related to this procedure. In the experimental part of the work, phenol-chloroform extraction is used. First of all, plasmids Channelrhodopsin-2, ASAP1 and Kir 2.1 were amplified in bacterial strain DH5. 22 isolations were accomplished and the yield was validated using gel electrophoresis and transfection to HEK293 cell line. The most successful isolation was the isolation of plasmid ASAP1, the overall percentage success rate was 30 %.
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Preparation of expression vectors for receptor NKp30 and its ligands B7-H6 and BAG-6
Pažický, Samuel ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
NK cells, the cells of non-adaptive imune system, are able to recognise viraly infected or oncogenous cells by many inhibiting and activating receptors that are expressed on their surface and eliminate them consequently. For NKp30, activating receptor of NK cells included in NCR (Natural Killer Cell Receptors) family, lately there were identified several ligands, including membrane protein B7-H6 expressed on oncogenous cells surface and BAG- 6, cell core protein with wide spectrum of functions. Aim of this thesis was preparation of expression vectors coding for receptor NKp30 and his ligands B7-H6 and BAG-6, enabling expression of these proteins in HEK293 cell line. Keywords: NK cell, plasmid, receptor, NKp30, B7-H6, BAG-6
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Colistin resistance in clinically important Enterobacteriaceae
Smělíková, Eva ; Tkadlec, Jan (advisor) ; Ježek, Petr (referee)
Colistin is a last-resort antibiotic used to treat serious infections caused by Enterobacteriaceae and other multidrug resistant gram-negative bacteria. Recently discovered plasmid-borne colistin resistance, mediated by the mcr genes, poses a serious risk to colistin therapy. The aim of this diploma thesis was to map the occurrence of Enterobacteriaceae carrying the mcr-1 to 8 genes in hospitalized patients, travellers, prospective colistin-resistant clinical isolates and in a retrospective collection of Enterobacteriaceae using a combination of selective cultivation and qPCR. Isolates with a detected mcr gene were characterized by Whole-Genome Sequencing. The localization of mcr genes was determined and other resistance genes and plasmids were identified. Furthermore, the physiological profile of selected colistin- resistant Escherichia coli isolates was characterized. In the presence of a subinhibitory amount of colistin, a strain carrying the mcr-1 gene may be favored. Later, the mcr-9 gene was described and its occurence was subsequently tested retrospectively. Enterobacter spp. isolates carrying the mcr-9 gene were mostly colistin-sensitive but, in some cases, resistance was induced after exposure to sublethal doses of colistin. The results of the study show that the incidence of plasmid-mediated...
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Knowledge and Opinions of Students on Genetically Modified Organisms
Semencová, Barbora ; Hlaváčová, Lucie (advisor) ; Vojíř, Karel (referee)
This diploma thesis is focused on topic of genetically modified organisms and their use in the practical sectors of human life. Theoretical part of the thesis defines general terms GMO, plasmid, genetic engineering, biotechnology. It also records historical milestones relating to the problematic, deals with individual techniques of genetic engineering and briefly states legislative procedures in context of dealing with GMO. It gives examples of transgenic organisms and summarizes advantages and disadvantages of their use.Practical part of the thesis contains educational program called "Genetically modified organisms", which was conceived by the author and includes a draft of a lesson inclusive of teaching materials - powerpoint presentations, worksheets, interactive worksheets, auxiliary text for teacher and written preparation. Research part deals with high school students change of view about using GMOs after completing the educational program. Due to analysis was proven that most of the attitudes and knowledge about GMO was changed after completing the educational program (for example in issues of willingness to consume GM food and animal products, perception of advantages and disadvantages etc.) Data was still unchanged in questions which cannot be affected by the program (control of food packaging or...
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Preparation of lentiviral expression vector with reporter gene
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Besides recombinant protein expression in prokaryotic cell lines (E. coli), systems, that could quickly, reliably and stably produce recombinant proteins in human cell lines, come to the fore. These cell lines assure proper tertiary structure and post-translational modification of the desired products. One of the ways to achieve production of recombinant proteins in human cell lines is the use of lentiviral vectors. This thesis describes the preparation of the lentiviral vector (plasmid) Daedalus, which contains a construct for recombinant expression of secreted alkaline phosphatase. For the preparation of the desired plasmid methods based on insertion of the secreted alkaline phosphatase gene using the restriction endonucleases and methods based on amplification by polymerase chain reaction (restriction-free cloning, transfer polymerase chain reaction and Gibson assembly) were used.
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Preparation of expression vectors for receptor NKp30 and its ligands B7-H6 and BAG-6
Pažický, Samuel ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
NK cells, the cells of non-adaptive imune system, are able to recognise viraly infected or oncogenous cells by many inhibiting and activating receptors that are expressed on their surface and eliminate them consequently. For NKp30, activating receptor of NK cells included in NCR (Natural Killer Cell Receptors) family, lately there were identified several ligands, including membrane protein B7-H6 expressed on oncogenous cells surface and BAG- 6, cell core protein with wide spectrum of functions. Aim of this thesis was preparation of expression vectors coding for receptor NKp30 and his ligands B7-H6 and BAG-6, enabling expression of these proteins in HEK293 cell line. Keywords: NK cell, plasmid, receptor, NKp30, B7-H6, BAG-6
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Preparation of expression vectors for NKp65 and KACL, new members of human NK cell receptor family
Mikulová, Barbora ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells create an important part of innate immune system. Their importance lies in their ability to recognize and kill abnormal cells, especially tumour cells and virally infected ones, without previous activation. To recognize their targets, NK cells use a wide variety of surface receptors, both activating and inhibitory. If a ligand binds to an NK receptor, immune response is triggered. Examples of such ligand-receptor pairs are NKp80-AICL and NKRP1-LLT1 on human lymphocytes. Another ligand-receptor system of this kind is NKp65 and KACL, two recently discovered lectin receptors on human immunocytes. KACL is the last and most recently characterized member of CLEC2 receptor family in humans. Its expression is almost exclusively restricted to skin. NKp65, a close relative of NKp80, is a glycoprotein which stimulates NK92MI cell cytotoxicity upon KACL engagement. NKp65 has been shown to bind to KACL with a fairly high affinity by surface plasmon resonance measurement. This thesis aims at describing the cloning of expression vectors coding for NK cell receptors NKp65 and KACL, expression of these proteins in HEK293T cell line and their purification. Keywords: NKp65, KACL, NK cell, lectin, receptor, plasmid (in Czech)
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