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Studying of Gene Expression Involved in Hyaluronic Acid Synthesis in Streptococcus Equi Subsp. Zooepidemicus Using DNA Microarrays and Real-Time PCR
Hrudíková, Radka ; Šeda,, Ondřej (referee) ; Bobek,, Jan (referee) ; Velebný, Vladimír (advisor)
Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.
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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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Účinky vybraných peptidů na jednobuněčnou řasu Chlamydomonas reinhardtii
Avdeeva, Olga
In the presented work, the modified novicidin peptides NVCAA and NVCHH were applied to the unicellular alga Chlamydomonas reinhardtii. The aim of the study was to determine the minimum inhibitory concentrations of the tested peptides on Escherichia coli bacteria with subsequent application on the unicellular algae Chlamydomonas reinhardtii. The assessment of the effect of the tested peptides on the algae was carried out using spectrophotometric analyses: chlorophyll A and chlorophyll B content, total flavonoid content, total antioxidant capacity and using the DPPH ASSAY method. Chlamydomonas reinhardtii molecular changes were determined by monitoring the amount of isolated RNA and the expression of APX1, APX2, CAT1, CAT2 genes using the Real-time PCR method. Statistically processed data showed that the application of NVCAA and NVCHH peptides induced considerable stress in the cilia.
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Sezónní dynamika šíření původce sazné nemoci kůry na lokalitě Brno - Sadová
Janovská, Kateřina
Sooty bark disease is caused by a parasitic fungus Cryptostroma corticale (Ellis & Ever-hart) Gregory & Waller, which survives in its host (mostly sycamore maple) in engophy-tic stage. After exposing the host to stress, especially drought stress, its strong pathoge-nic effects prevailes, which may be fatal for the host. At the research area in Soběšice, aerobiological sampling took place from spring to autumn using automatic volumetric spore trap. DNA extraction and real-time PCR were used to determine the number of C. corticale spores in the samples. The effect of meteorological factors on the spore amounts was determined. More spores were observed in the spring months than in the autumn months under similar conditions.
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The influence of storage time on authentication of plant-based foods
Trödlerová, Martina ; Šislerová, Lucie (referee) ; Fialová, Lenka (advisor)
Fruit baby foods and purees belong among adulterated food, due to the prices of materials for their production. More expensive ingredients are replaced by cheaper, lower quality ones. This bachelor thesis deals with influence of storage time on the determination of samples authenticity The theoretical part of the thesis deals with the chemical composition of fruit (especially apples and peaches), production technology and methods of determining authenticity. In the experimental part, model samples were prepared and DNA was isolated from them. Then, using two different primer pairs, this DNA was amplified by real-time PCR and a specific product was detected by analyzing melting curves. On gel electrophoresis, it was possible to confirm the presence of these specific products. The last method was HPLC, where phenolic substances were analyzed.
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Alternative beverages of probiotic character
Moravec, Štěpán ; Langová, Denisa (referee) ; Trachtová, Štěpánka (advisor)
Bachelor thesis deals with explanation of therm probiotics, properties of each probiotic bacteria genuses. preparation of coffee and tea, and also deals with probiotic coffee and tea and molekular diagostic methods which were used during bachelor thesis. There were used mikrobiological and molecular diagnostic methods for analysis of probiotic coffee and teas. Namely for confirmation of bacterial genus Bacillus and for confirmation of survavibility of Bacillus during high temperatures. Presence of bacterial DNA in samples of coffee and teas was confirmed by conventional PCR method and by real-time PCR method. Survibility of genus Bacillus during high temperaturewas confirmed by PMA-PCR method.
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New trends in the use of probiotics in cosmetic products and their characterization
Dvořáková, Monika ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
Probiotic products have always been, in the form of various dairy products, a part of our diet. In recent years, they also became prominent in the pharmaceutical sector, where they can often be found as food supplements, and in the cosmetic sector, where they are being added to intimate hygiene and skin care products. The first part of the thesis consists of conducting a theoretical research in the field of probiotics, microbiome and other possibilities of where we can find probiotic organisms in cosmetic products. Further, a part of the thesis is devoted to methods of cultivating and isolating bacterial DNA from the following products: Benton, Mechnikov, La Rosche-Posay creams and Benton and Neogen masks, in sufficient quality for subsequent PCR amplification. DNA was isolated using magnetic carriers, a commercial kit, and phenol extraction. The presence of bacterial DNA was proved by a q-PCR analysis using genus-specified primers of selected bacteria. Genus identification corresponded with the information declared by the manufacturer. To conclude, a PCR and propidium monoazide dye were used in combination for quantification of viable and dead cells, and a HRM analysis was performed.
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Use of advanced molecular biology techniques for detailed characterization of probiotic bacteria from a dietary supplement
Folwarczná, Tereza ; Trachtová, Štěpánka (referee) ; Smetana, Jan (advisor)
The aim of the diploma thesis was the identification of probiotic bacteria from a probiotic food supplement in the form of syrup. DNA was isolated from the complex matrix of the product in sufficient purity and quality suitable for the real-time PCR method followed by PCR-HRM. Specific primers for the genera Lactobacillus, Bifidobacterium and Streptococcus and at the species level for the species Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus thermophilus were used for amplification by real-time PCR and PCR-HRM. After optimizing the conditions for specific primers, all declared bacteria were detected in the product.
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