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Optimization of plasmid DNA isolation by magnetic particles
Chlopková, Barbora ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
The theoretical part summarizes information on the isolation and purification of plasmid DNA and nucleic acids as. Plasmid DNA is often used in gene engineering as a vector for the transfer of a particular gene. Its insulation and transportation in sufficient quality is crucial for other processes associated with it. Isolation and survival of pDNA using magnetic carriers of different concentrations of PEG 8000 in combination with 1M NaCl was investigated in experimental parts. Furthermore, the isolation of pDNA using commercial kits was examined.
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The effects of chemicals on cell lines viability
Zemanová, Anita ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
The subject of this diploma thesis is the influence of selected chemicals on cell lines viability. The theoretical part contains options of cancer treatment by using chemotherapeutics including their mechanism of action and side effects. Additionally, there are described alternative DNA structures with focus on G-quadruplexes and ligands that interact with G-quadruplexes. These compounds are promising drugs in cancer treatment due to their high specificity to G-quadruplexes, which are found in telomeres of chromosomes. G-quadruplex interacting ligands by stabilization of G-quadruplexes can inhibit the enzyme telomerase, which is necessary for telomere lengthening of rapidly dividing cancer cells. Additionally, the possibilities of viability assays are summarized in the theoretical part. The aim of the experimental part was comparing cytotoxic activity between commercially available chemotherapeutics and selected G-quadruplex interacting ligands. Another task was the study of apoptosis and necrosis after the treatment of selected chemicals on cell lines and after the localization of ligands interacting with G-quadruplexes in the cells of the breast cancer cell line. In the experimental part, G-quadruplex interacting ligands have been shown to exhibit similar cytotoxic activity to commercially available chemotherapeutic agents.
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Preparation and expression of p53 protein isoforms using the GATEWAY expression system
Wikarská, Monika ; Hrstka, Miroslav (referee) ; Brázda, Václav (advisor)
The TP53 gene can express protein p53 and 11 another isoform proteins N- and/or C-terminally truncated by using two promoters and alternative splicing. The p53 isoforms are found in both healthy and tumorous tissues, and are intensively studied in relation to cancer diagnosis, prognosis and treatment. In this work, the p53 isoforms were subcloned into expression vectors by LR reaction adapted from Gateway cloning system. The expression vectors were designed for protein production by bacteria E. coli strain BL-21. The constructs containing p53 isoforms were encoded together with two fusion proteins, glutathione-S-transferase and polyhistidine tag under the control of the same promotor for the affinity chromatography protein isolation. All the clones underwent Sanger sequencing for verification after homologous recombination. Sequencing confirmed the accuracy of the subcloned isoforms p53, 133p53, 160p53, p53 and 160p53 into an expression vector pDEST15-N6xHis-GST-GW-DEST. Protein 160p53 was expressed in BL-21 and isolated using both HIS and GST tag interacion. Isolation using HIS tag yielded in a higher protein concentration then the isolation mediated by the interaction of the glutathione-S-transferase.
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Use of high resolution melting analysis for the study of lactic acid bacteria
Knápková, Monika ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.
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Using different methods of DNA isolation of lactic acid bacteria in molecular biological methods
Chvalkovská, Eva ; Skoumalová, Petra (referee) ; Brázda, Václav (advisor)
This thesis focused on the probiotic bacteria, DNA isolated from these bacteria by three different methods and the effect of isolation on DNA identification using molecular biological methods. Probiotic bacteria are an important part of human intestinal tract. They have an important role in the function of the immune system due to adhesion to the mucosa of the intestinal flora. They create a inhostile environment for pathogens. Probiotic bacteria are commonly taken in the food like dairy products or food supplements. However, overuse of antibiotics is at risk of passing on the intrinsic resistance that probiotic bacteria have to the pathogenic bacteria. The intrinsic resistence they have to maintain the natural homeostasis of the intestinal tract. It is important to effectively identify risky probiotic bacteria that have the ability to transmit resistance to eliminate their presence in food and dietary supplements. Three methods of DNA isolation like phenol extraction method, magnetic particle isolation and commercial kit isolation were used in the experimental part. DNA was isolated from three dietary supplements, namely Biopron 9 premium, Linex forte and GS Lactobacily forte 21. The purity and concentration of the isolated DNA was detected spectrophotometrically. The presence of individual DNA strains in dietary supplements was confirmed by real-time polymerase chain reaction. The best method of isolation in terms of purity and concentration of isolated DNA was evaluated by RT-PCR and spectrophotometry using a commercial kit isolation method.
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Screening of biotechnological potential of selected members of the genus Geobacillus and other related genuses
Kouřilová, Xenie ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.
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Preparation of DNA with local DNA structures
Lofítková, Ellen ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
In this thesis I focused on quadruplexes and cruciforms formed by DNA. Plasmids pBluescript and others derived from it by inserting oligonucleotid sequences were studied. Sequences forming quadruplexes and cruciforms were found by in silico analysis by QGRS Mapper and Palindrome analyser. Plasmids were transformed into E. coli, isolated and then cleaved with enzymes S1 nuclease and restriction endonuclease ScaI. Cleaving with S1 nuclease predicated presence of local structure. Combined S1/ScaI cleavage did not bring satisfying results due to lost of DNA during purification.
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Impact of temperature and drought on gliadins content in two varieties of wheat
Seidlová, Kateřina ; Brázda, Václav (referee) ; Hrstka, Miroslav (advisor)
This bachelor’s thesis focuses on the effect of high temperature and drought on protein content of gliadin fraction on two varieties of wheat. Chosen varieties were Hyfi and Julie, cultivated at 26, 29, 32, 35 and 38 °C during flowering in watering controlled conditions. The condition for ‘wet’ samples was at least 70 % soil moisture and for ‘dry’ samples less than 30 % soil moisture. After harvesting, the seeds were milled into flour from which the gliadins were extracted with 2-chlorethanol. A-PAGE method was used for gliadin separation, quantification was carried out through computer densitometry. A significant genotype effect was discovered. Whilst temperature ranging from 26-38 °C with simultaneous drought stress had no significant effect on gliadin content of Hyfi variation, gliadin content of Julie variation shown obvious maximum at 32 °C. Therefore, Hyfi variation shown better resistance to heat stress than Julie variation. Both variations had higher gliadin content under drought stress than under good watering conditions.
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Preparation of constructs for protein isolation and its testing
Osadchuk, Olha ; Kostovová, Iveta (referee) ; Brázda, Václav (advisor)
This study is focused on describing of recombinant protein production. Protein p53 was chosen, as one of the most important tumor suppressor proteins, for studying this issue. The p53 protein is responsible for the gene regulation, control of cell cycle and DNA replication. P53 is the most mutated gene in human cancer. Several point mutations of p53 protein was chosen for work with. The theoretical part describes main properties of protein, expression systems, Gateway cloning system and methods of protein purification. In the experimental part are described the procedures of preparing of the expression vectors by Gateway technology, cell transformation and DNA plasmid isolation. Using cloning technology were prepared three expression clones, they were transformed into competent cells and after was done DNA isolation.
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Optimization of DNA isolation form yogurt cultures and their detection by RT-PCR
Šurková, Alice ; Němcová, Andrea (referee) ; Brázda, Václav (advisor)
The thesis has optimized DNA isolation from pure yoghurt cultures and yoghurt products. The isolated DNA was than subjected to RT-PCR analysis. In the first part of the thesis, DNA isolation from pure yoghurt cultures using a commercial kit was evaluated as more effective than isolation by phenol extraction and magnetic microparticles. To assess the quality and quantity of DNA obtained the spectrophotometric determination of concentration and purity and qPCR were used. DNA of a total of ten pure yoghurt cultures in a quality suitable for PCR was obtained using the commercial kit. In the second part of the thesis, bacterial DNA was isolated from yoghurt products using the same commercial kit with a previous sample washing by lysation solution. DNA of six yoghurt products was isolated this way. Furthermore, two packages of homemade yoghurt were mad of each product, of which DNA was isolated in the same way. DNA obtained from yoghurts was subjected to RT-PCR using six pairs of primers (V3_F a V3_R, V6_F a V6_R, V1_F a V1_R, GroHRM_F a GroHRM_R, UPF a UPR, P1V1 a P2V1) and using the pure cultures DNA as a positive controls. The results confirmed the presence of cultures declared in each yoghurt and their ability to multiply after inoculation into a new medium (milk).
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