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Creating and testing bioluminescence sensors for cytokinins in Arabidopsis thaliana
Novotná, Lenka ; Petrášek, Jan (advisor) ; Vosolsobě, Stanislav (referee)
Advances in microscopy and the sensitivity of novel biosensors have enabled the development of new methods for monitoring phytohormones with low invasiveness and relatively high resolution, providing greater insight into phytohormone function in plants. Genetically encoded indirect biosensors for cytokinins, one of the main plant hormones, are widely used for the tracking of dynamic changes in their levels during plant development and growth. The cytokinin-sensitive synthetic promoters used as cytokinin biosensors are being continuously improved, providing better insight into cytokinin function. However, the reporter systems used for the visualizations are either based on the enzymatic reaction producing colour or on the usage of fluorescence proteins. Luminescence-based biosensors are often used to visualize signaling outputs of plant hormones, but they are based on exogenous addition of luciferin substrate. Therefore, in this thesis, I have introduced a new technique for the generation of cytokinin signaling output sensors based on autonomous bioluminescence. This autonomous bioluminescence represents a non-invasive technique that depends on the expression of the whole autonomous bioluminescence system from fungi Neonothopanus nambi, in particular, the caffeic acid cycle with the fungal...
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Bioluminescence in Dinoflagellates
Zahradníček, Ondřej ; Němcová, Yvonne (advisor) ; Pichrtová, Martina (referee)
Bioluminescence is a relatively widespread phenomenon in nature. A variety of organisms have the ability to emit light. The aim of this bachelor thesis is to summarize the latest findings on bioluminescence in dinoflagellates. These organisms are the most important protist lineage capable of producing light flashes. Indeed, they are responsible for most of the bioluminescence observed in surface the seas and oceans. This bachelor thesis discusses the biochemical reaction of bioluminescence in which the substrate (luciferin) is oxidized in the presence of the enzyme luciferase. The thesis introduces the major species of luminescent dinoflagellates that frequently cause toxic harmful algal blooms. Then the factors affecting the intensity of bioluminescence are discussed. Last but not least, dinoflagellates can use bioluminescence as protection from predators. For example, they may use a flash of light to startle predators or use the flash as a "burglar alarm" to attract predators of their predators. Key words: bioluminescence, luciferin, luciferase, dinoflagellates, light flash
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Porovnání promotorů pro využití v klíštěcím (\kur{Ixodes}) expresním systému
MUSTACOVÁ, Johana
Gene manipulation can be a convenient tool for tick control, however functional procedure for gene manipulation of ticks was not determined yet. The creation of plasmid vectors with high ability of gene expression driven by functional promoters is crucial for genetically modified ticks. To investigate effective tools for tick genes manipulation within the scope of this master thesis, transfection procedures for various types of Ixodes tick cell lines were optimized. As well as expression vectors for use in tick cells were tested. For this purpose, expression plasmid vectors containing luciferase reporter genes driven by eukaryotic and viral promoters were used.
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Preparation and validation of a system for the study of regulation of gene expression of yeast linear cytoplasmic plasmids
Horáčková, Kamila ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
There is currently very few information about the transaltion of linear cytoplasmatic plasmids occured in yeast cells Kluyveromyces lactis. However, there is a relatively well developer information about their transcription apparatus. A study of transkript linear plasmids revealed an atypical organization at the 5ʼ end. Those ends contain nontemplate polyadenylation and they are missing the N7 methylguanosine hat. Because of the presence of this structure, which is localized at 5ʼend of plasmids specific mRNA, raised a question regarding the iniciation of the translation. The present thesis is focused on the preparation of reporter systém suitable for studying the influence of a number of the nontemplate adenosins, which were added at the 5ʼ ends of mRNA linear plasmids. The frist step was making a construction of dual yeast cell plasmids carring two reporters genes, which are under the controle of two different promoters. After a successfull construction, the aktivity of promoters TEF1 and PGK1 was measured, whereby the promoter TEF1 proved twice stronger. The transcription start site of both promotor was determined. The second step was the construction of a reporter system directly in yeast cell plasmid pGKL. Reporter genes were under the controle of two promoters originating from the pGKL...
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