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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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Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell line
Jablonská, Dominika ; Čmiel, Vratislav (referee) ; Svoboda, Ondřej (advisor)
This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.
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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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Electrophysiological characterization of Kir2.1 membrane channel
Měsíčková, Klára ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.
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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Skala, Kateřina ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
DNA isolation is one of the basic methods in molecular biology. There are several methods of DNA amplification and isolation. In this paper phenol-chloroform extraction of three plasmid types - Channelrhodopsin-2, ASAP1 and Kir 2.1 is used. Six plasmids were isolated in total. These plasmids are then validated using gel electrophoresis. Successfully isolated plasmids are then transfected to HEK293 cells and images taken on confocal microscope 24, 48 and 72 hours after transfection.
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Application of tissue culture test plates for production of recombinant protein in HEK293 cells; determination of optimal conditions
Krzyžanková, Marcela ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Efficient production of the recombinant proteins (r-proteins) must be based on previous testing of an expression of a small amount of the r-proteins. This work focuses on optimizing the expression of the r-proteins in 12-well plates. It includes testing of an appropriate speed of shaking, production and transfection volume. It compares all the current testing vessels (it compares a 50-ml centrifugation tube to new tested plates that can substitute the unsuitable tubes). It also compares these new tested plates to production square bottles in order to compare the r-protein expression in the plates to the r-protein expression in the bottles. It monitors effects of carbon dioxide on a number of vital cells, their viability, a relative frequency of positive cells on GFP in various cultivation vessels (plates, tubes, bottles), and pH of HEK 293 cellular cultivation during the 4-day cultivation process as well. On the basis of the results and statistical processing of the results, we have set the optimal agitation speed of 230 rpm for the 12-well plates. We have also set the appropriate production and transfection volume of 2 and 0.5 ml for the 12-well plates. In order to evaluate variables and compare cultivations in all the vessels, the tubes could be substituted by the plates. There is a statistically significant impact of carbon dioxide on the number of cells, their variability, relative frequency of cells (positive on GFP) and pH of the cellular HEK 293 cultivation in the cultivation vessels. There is the strongest r-protein expression in carbon dioxide conditions. The results of this work allow to employ the 12-well plates when we aim to test the expression of the r-proteins in a small amount and in carbon dioxide conditions. On the basis of the findings, the expression of the r-proteins in the 12-well plates and carbon dioxide conditions can substitute the expression of the r-proteins in the production bottle and in carbon dioxide free conditions.
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Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell line
Sanetrníková, Dominika ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.
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Effects of trypsin to HEK293 cell membrane
Lipnická, Soňa ; Provazník, Ivo (referee) ; Čmiel, Vratislav (advisor)
This Bachelor’s thesis examines the effect of trypsin on the membranes of cell cultures. HEK293 cell lines nontransfected and cell lines stably transfected through the CaV3.1 membrane channels were selected for the optimization. They were gradually exposed to 0.25, 0.375 and 0.5 % trypsin. The concentration of trypsin influences the separation of the adherent cells on structure, as well as the degradation of cell membranes, which influences the cell cultures viability. Part of this thesis is an attempt to establish a methodology for the research of the effect of trypsin on membranes of HEK293 cell lines.
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Preparation of growth factor TGF-β3 with coiled-coil tag
Otépka, Tomáš ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Growth factors represent a group of significant substances in the metabolism of organisms. They are signaling molecules that control cell activity at the endocrine, paracrine, or autocrine levels. They act as key mediators binding to cell receptors, triggering a cascade of reactions leading to the regulation of genetic transcription in the cell nucleus and stimulation of cellular response. Growth factors influence various physiological functions such as cell proliferation, cell differentiation, and tissue healing. The utilization of growth factors is evident, for example, in regenerative medicine. For a similar purpose, research has been initiated to prepare the growth factor TGF-β3 with the possibility of attaching it to a polymeric carrier using a coiled-coil tag. This work focuses on the recombinant production of TGF-β3 - its analog with a latency-associated peptide (LAP) - and the application of techniques applicable to the intention of this work, specifically, attaching the protein to a polymeric carrier based on amino acids. Given the structural complexity with which growth factors are physiologically released from cells, the preparation of growth factors with a coiled-coil tag in vitro represents an unexplored challenge in the field of recombinant protein expression. In our HEK293T cell line...
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Preparation of the high-affinity binding domain of protein B7-H6
Jeníček, Jakub ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
Natural killer cells are part of innate immunity and play a key role in defending the organism. Their role in the defense against tumors and anti-tumor therapy has been the subject of multiple research projects because tumors are among the most frequent causes of death worldwide. Tumor therapy is often complicated and invasive; therefore, finding new therapeutic approaches that target naturally occurring defense mechanisms is advantageous. One of the key mechanisms used by NK cells to recognize tumor cells and eliminate them is signaling via their receptor NKp30. The binding of an activation ligand to this receptor can induce the activation of a cytotoxic response, leading to the elimination of the tumor cell. One of the activating ligands that can bind to NKp30 is B7-H6, a cell surface protein found on certain types of tumors. However, the interaction between B7-H6 and NKp30 has not been wholly described yet. This thesis focuses on different methods, which can be used for obtaining the B7-H6 domain bearing a fluorescent label, that could be used to visualize NKp30 on the cell surface, thus allowing for further description of the interaction between these molecules. KEY WORDS NK cells, NKp30, B7-H6, sortase A, HEK293
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