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Transient transfection of a serum free cell culture using polyethyleneimines
Čutová, Michaela ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Master’s thesis deals with the transient transfection of the serum free animal cell culture using polyethyleneimines. In the theoretical part formation of recombinant DNA molecules, used expresion vectores, used DNA transfer and detection of recombinant proteins are discussed. The experimental part deals with efficiency of the polyethylenimine mediated transient transfection under various experimental conditions. 293HEK/EBNA cell line was chosen as an experimental model. First the most effective plasmide - pCEP4/SEAP was selected. Then three transfection methodes were tested: Muller (2005), Durocher et al. (2007) and Backliwal et al. (2008). The highest recombinant protein expresion was reached using the method of Backliwal et al. (2008).
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The safety of probiotics used in foods
Balažovičová, Nikola ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The theoretical part of this thesis focuses on definition of probiotics, their health benefits, probiotic foods, safety criteria for probiotics used in foods, including methods for the identification of probiotic bacteria. Experimental part of work was focused on the identification of probiotic bacteria contained in the milk product Actimel. DNA quality suitable for the PCR was isolated by magnetic microparticles P(HEMA-co-GMA). Identification was estimated using the method of polymerase chain reaction. The presence of DNA of domain Bacteria, genus Lactobacillus and species Lactobacillus casei was confirmed by gel electrophoresis of PCR products. Specific PCR products of the sizes of 466 bp (domain Bacteria), 250 bp (genus Lactobacillus) and 136 bp (Lactobacillus casei) were detected after amplification of DNA isolated from a probiotic milk product.
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Development of transient transfection protocol for HEK293 EBNA1 cells
Šmíd, Jiří ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.
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Molecular identification of selected species of lactic acid bacteria and bifidobacteria in food additives
Riegelová, Kristýna ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria and bifidobacteria are natural part of microflora of gastrointestinal tract. In the present, day they are grossly exploited in food processing industry. The aim of the work was molecular identification of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of two food additives. Total DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified in genus-specific and species-specific PCRs. Amplicons were detected by agarose gel electrophoresis. Results were compared with declared specification given by producers in three different batches.
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Monitoring the success of transfection of cell line 293 HEK
Dvořák, Tomáš ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Diploma thesis is based on monitoring the succes of transfection of cell linie HEK293. In theoretical part are described principles of transfection methods, cell lines, vectors and reporter genes. HEK293 cells EBNA1 were used for practical part. It was studied the difference between GFP and EGFP plasmids. As well as using various transfection reagents under different culture conditions.
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Probiotic bacteria and authenticity of milk products
Storozhko, Viktoriya ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotics are living microorganisms that have positive effects on human health after consumption. The theoretical part of the bachelor thesis describes properties and health effects of probiotics and their use in food industry. The experimental part was focused on the isolation of PCR-ready DNA from two dairy probiotic products. The presence of target bacterial DNA was confirmed using PCR methods.
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Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
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Nucleic acids as terapeutic agent
Ráčková, Lucie ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of molecular biology development of oncotherapy proceeds. The major progress of modern medicine is gene therapy. In the gene therapy are two categeories, namely, viral vectors and nonviral vectors which are used mainly. Nonviral vectors include plasmids. Plasmid DNA used in medicine must be perfectly purified. Chromatographic methods are mainly used at present. Research and development deals with other methods for example two-phase aqueous systems and magnetic carriers. In experimental part of this thesis, isolation of pUC 19 plasmid DNA from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Quality of isolated plasmid DNA was verified spectrophotometrically and by agarose gel electrophoresis. Isolated plasmid DNA was purified using three methods: RNA in plasmid DNA was precipitated by lithium chloride, RNA was degraded by immobilized RNase A and plasmid DNA was purified using two-phase aqueous system.
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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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