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Evolutionary and genetic engineering of bacterial producers of polyhydroxyalkanoates
Nováčková, Ivana ; Patáková, Petra (referee) ; Koutný, Marek (referee) ; Obruča, Stanislav (advisor)
This doctoral thesis deals with the topic of evolutionary and genetic engineering of polyhydroxyalkanoates (PHA) producing bacteria. Apart from these topics, the issue of biotechnological production of PHA on model hydrolysates of lignocellulosic biomass with the use of extremophilic microorganisms is also studied, as well as the development of an alternative method of PHA isolation. The themes were freely linked to previous experiments and reflected the currently solved projects in a working group. Doctoral thesis is prepared in the form of a commented discussion of published works, which are part of it in the form of appendices. Evolutionary engineering was mainly applied to the model PHA producing bacterial strain Cupriavidus necator H16. By adaptation to levulinic acid, isolates producing copolymer P(3HB-co-3HV) with a higher content of the 3HV fraction were obtained, which leads to improved properties of the polymer for further processing. As well as culture growth also the amount of total PHA in the biomass was higher. By long-term adaptation of the same strain to osmotic stress and the presence of copper ions, the isolates which are characterized in the second publication, were obtained. Based on obtained data, it was possible to observe differences in the adaptation process, where the adaptation to osmotic stress was gradual, while a significant step in the increase of biomass and PHA signaling faster adaptation was observed for copper. Based on the analyses, the significant role of PHA in the adaptation of the C. necator H16 strain to the tested stressors was discussed, it did not consist only in the increase in the amount of polymer in the biomass, but also in enhancement of whole PHA cycle, which also leads to an increase of the pool of monomeric units showing protective functions. By adaptation to -captolactone, a unique precursor of 4HB, the copolymer P(3HB-co-4HB) was obtained. The properties of this copolymer are again more favorable than of the homopolymer P(3HB), even with a low content of 4HB, which we also achieved in a laboratory bioreactor. A further increase in the 4HB fraction could be achieved using deletion mutants with the absence of relevant genes, which is discussed more in the text. The production of PHA on models of lignocellulosic biomass hydrolysates originating from, for example, the food industry was tested in combination with the use of extremophile producers, when the preference of the contained monosaccharides (hexoses, pentoses) for individual producers was discussed. For the purpose to get closer to real hydrolysates, the resistance of the strains to relevant potential microbial inhibitors was also tested. The susceptibility of halophilic and thermophilic PHA producers to osmotic stress was used in the development of an alternative isolation approach that would reduce the economic and ecological burden of the process compared to standard extraction using chlorinated solvents. Application of SDS detergent at low concentrations while simultaneously exposing the cells to higher temperatures led to the gain of high purity polymer without loss of yield. The recycling process of used SDS is also a possibility.
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PCR Detection of GMO in Food and Feed
ŠKRNOVÁ, Dominika
The term "genetically modified organism (GMO)" has become a controversial topic in many countries as its benefits for both food producers and consumers are accompanied by potential biomedical risks and environmental side effects. In this thesis, I attempt to summarize up-to-date knowledge about genetically modified (GM) crops. I also introduce different methods for detecting GMOs in food and feed. In the practical part of this thesis, thirty food and feed samples containing maize were randomly collected from different sources. The aim of this study was to test these samples for the presence of GMO, verify the information given on the label, and check that it complies with the EU legislation requiring that any food or feed containing more than 0.9% GM content has to be labeled. The next aim was to optimize the method for DNA extraction from the tested samples. In order to isolate genomic DNA from the samples, I used the NucleoSpin Food kit and the MagCore Automated Nucleic Acid Extractor. The concentration of extracted DNA was evaluated by ultraviolet (UV) absorption using a BioSpec-nano spectrophotometer. The gene coding for the zein storage protein of maize was used to confirm the presence of amplifiable maize DNA. DNA extraction was followed by PCR protocols using different sets of primers to detect four maize events: Bt11, Bt176, Mon810, and T25. Agarose gel electrophoresis was used for the separation of DNA fragments that were subsequently visualized using InGenius manual gel documentation system. In spite of the curent regulations, none of the three samples tested positive for the presence of MON810 maize had any such indication on their label. The samples that claimed to be "GMO free" on their label actually did not show the presence of any of the GM maize events tested.
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Potential of genetic engineering for breeding plants tolerant to abiotic stresses: cold resistance in rice
Lotová, Gabriela ; Lipavská, Helena (advisor) ; Janská, Anna (referee)
With increasing population and climate change, there has been an increase in efforts to breed more efficient crops. Genetic engineering has opened unprecedented breeding possibilities in developing plants with desired traits. Transgenic crops with better qualities, including resistance to adverse environmental conditions, can contribute to solving problems of hunger and malnutrition in developing countries. Although society perceives genetically modified crops rather negatively, these crops are widely used as feed for livestock and outside Europe also for human nutrition. Because of the complexity of resistance to abiotic stress, the utility of genetic manipulations for the breeding of resistant plants was previously not anticipated. However, it turned out that modification of the stress signalling cascade or transcription factors can lead to success. This thesis summarizes the possibilities of genetic modification of crops, which may result in better tolerance to cold, and is mainly focused on rice. Part of the work deals with transduction of cold signal, whose modification can also result in increased tolerance to cold. Another part deals with transcription factors that activate expression of low temperature- resistant genes. The greatest attention is paid to CBF/DREB transcription factors that...
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Genetically modified plants in practical applications
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Cvrčková, Fatima (referee)
Genetic engineering (GI) of plants is a very current topic, and more and more controversial, since it is becoming an inseparable part of our lives. GI has, among other things, a great potential to help solve the current problem of hunger and malnutrition in certain parts of the world. The goal of this project is to clarify what genetically modified (GM) plants are, to present the possibilities of their practical use, to explain methods of preparation and to consider their advantages and eventual risks. By GM plant we understand a plant whose genetic information has been changed by introducing or removing part of the genetic information (a section of DNA, generally a gene). The most frequently used methods of transformation are the transformation by Agrobacterium tumefaciens and the balistic method. GM plants can be used for production of food or feed in better quality and greater quantity, thanks to an introduction of higher resistance, whether it is to biotic stress (pest, virus or bacterial resistance) or to abiotic stress (drought, salinity, toxic substance in the ground..). In particular, the resistance to abiotic stress is an important issue these days, since through the climate changes some regions suffer more and more from insufficient precipitation and consequent drought. Some of the GM plants grown...
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Genome editing using programmable endonucleases
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Sýkora, Michal (referee)
Programmable endonucleases are engineered proteins that recognize specific nucleotide sequences and that are capable of introducing double-strand breaks within these sequences. Zinc-finger nucleases have been used extensively as a tool in genome editing, the practice of introducing changes into genomes of cell lines or whole organisms as a way to study gene function. Recently, new types of programmable endonucleases have emerged in the form of transcription activator like effector (TALE) nucleases and the CRISPR/Cas system. The types differ in respect to their mechanism of function, accessibility, selectivity, frequency of off-target cleavage and cytotoxic effects. Here, we compare zinc-finger nucleases, TALENs and the CRISPR/Cas system and explore their current and possible future applications in a broad spectrum of research ranging from developing genetically modified organisms to gene therapy. Powered by TCPDF (www.tcpdf.org)
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Science Fiction and the Prediction of Technology, Past and Present
Skorobogatova, Margarita ; Sedláček, Pavel (referee) ; Froehling, Kenneth (advisor)
Cílem této bakalářské práce je prozkoumat, zda-li sci-fi dokáže predikovat budoucí vývoj technologií. První kapitola představuje sci-fi spisovatele 19. a 20. století, kteří předpovídali řadu technologických vynálezů vyskytujících se v současnosti. Druhá kapitola se zabývá sci-fi kinematografií, která je popsána a doložena příklady z sci-fi filmů a televizních seriálů. Poslední kapitola je základem různorodých technologických vynálezů současnosti, které jsou popsány a demonstrovány režiséry sci-fi filmů, a považovány za nejvíce pravděpodobné přiklady, které můžou být realizovány v blízké budoucnosti. Závěr popisuje výsledky provedených výzkumů a definuje případný vývoj v budoucnu.
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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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Birth
Piskova, Olena ; Klímová, Barbora (referee) ; Gabriel, Michal (advisor)
The thesis is a continuation of my subject, "Why?", Also includes the theme of man and the environment. I started to be interested in the controversial topic of genetically modified organisms, genetic engineering. I found a personal story in it. Now it is difficult to comment on the extent to which these researches are important, but we shouldn’t stop there.
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PCR detection of genetically modified soybean in food and feed
CHADIMOVÁ, Nikola
Genetically modified organisms which genetic equipment has been specifically targeted of have been acquiring properties they did not have before. One of the main reasons for this aimed genetic modification in plants is herbicide resistance. Especially in soy case is used the modification to resist to glyphosate. The safety of the food which has been made of genetically modified crops or products of animal fed with GM feed has not been proved so far. This bachelor thesis focuses on methods for detection of GMOs in food and feed. Also, it focuses on verifying the data concerning the presence of GMOs presented by companies on food or feed packaging. The research part of this work contains information about genetic engineering, the issue of genetically modified organisms and also focuses on the description of techniques needed to detect GMOs, especially in processed foods, but also in feeds available on the Czech market. The methodical part of this bachelor thesis focuses on the detection of CaMV 35S promoter occurring in DNA crops that have undergone genetic modification. The presence of a specific transgenic material involved the detection of a particular glyphosate-resistant gene, which is particularly known under the trade name Roundup. There were used basic molecular biological methods to analyze GMOs.
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Potential of genetic engineering for breeding plants tolerant to abiotic stresses: cold resistance in rice
Lotová, Gabriela ; Lipavská, Helena (advisor) ; Janská, Anna (referee)
With increasing population and climate change, there has been an increase in efforts to breed more efficient crops. Genetic engineering has opened unprecedented breeding possibilities in developing plants with desired traits. Transgenic crops with better qualities, including resistance to adverse environmental conditions, can contribute to solving problems of hunger and malnutrition in developing countries. Although society perceives genetically modified crops rather negatively, these crops are widely used as feed for livestock and outside Europe also for human nutrition. Because of the complexity of resistance to abiotic stress, the utility of genetic manipulations for the breeding of resistant plants was previously not anticipated. However, it turned out that modification of the stress signalling cascade or transcription factors can lead to success. This thesis summarizes the possibilities of genetic modification of crops, which may result in better tolerance to cold, and is mainly focused on rice. Part of the work deals with transduction of cold signal, whose modification can also result in increased tolerance to cold. Another part deals with transcription factors that activate expression of low temperature- resistant genes. The greatest attention is paid to CBF/DREB transcription factors that...
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