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Preparation and purification of plasmid DNAs
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of therapeutic methods in medicine, like are DNA vaccines and gene therapy increases demand for new processes for the isolation and purification highly pure plasmid DNA. Most often used methods of purification plasmid DNA are chromatographic methods. In experimental part of this thesis was performed isolation of plasmid pUC-19 DNA plasmid via alkalyne lysis. And purification of plasmid was performed by liquid chromatography and agarose gel electrophoresis.
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Colistin resistance in clinically important Enterobacteriaceae
Smělíková, Eva ; Tkadlec, Jan (advisor) ; Ježek, Petr (referee)
Colistin is a last-resort antibiotic used to treat serious infections caused by Enterobacteriaceae and other multidrug resistant gram-negative bacteria. Recently discovered plasmid-borne colistin resistance, mediated by the mcr genes, poses a serious risk to colistin therapy. The aim of this diploma thesis was to map the occurrence of Enterobacteriaceae carrying the mcr-1 to 8 genes in hospitalized patients, travellers, prospective colistin-resistant clinical isolates and in a retrospective collection of Enterobacteriaceae using a combination of selective cultivation and qPCR. Isolates with a detected mcr gene were characterized by Whole-Genome Sequencing. The localization of mcr genes was determined and other resistance genes and plasmids were identified. Furthermore, the physiological profile of selected colistin- resistant Escherichia coli isolates was characterized. In the presence of a subinhibitory amount of colistin, a strain carrying the mcr-1 gene may be favored. Later, the mcr-9 gene was described and its occurence was subsequently tested retrospectively. Enterobacter spp. isolates carrying the mcr-9 gene were mostly colistin-sensitive but, in some cases, resistance was induced after exposure to sublethal doses of colistin. The results of the study show that the incidence of plasmid-mediated...
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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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Preparation of lentiviral expression vector with reporter gene
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Besides recombinant protein expression in prokaryotic cell lines (E. coli), systems, that could quickly, reliably and stably produce recombinant proteins in human cell lines, come to the fore. These cell lines assure proper tertiary structure and post-translational modification of the desired products. One of the ways to achieve production of recombinant proteins in human cell lines is the use of lentiviral vectors. This thesis describes the preparation of the lentiviral vector (plasmid) Daedalus, which contains a construct for recombinant expression of secreted alkaline phosphatase. For the preparation of the desired plasmid methods based on insertion of the secreted alkaline phosphatase gene using the restriction endonucleases and methods based on amplification by polymerase chain reaction (restriction-free cloning, transfer polymerase chain reaction and Gibson assembly) were used.
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Preparation of expression vectors for NKp65 and KACL, new members of human NK cell receptor family
Mikulová, Barbora ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells create an important part of innate immune system. Their importance lies in their ability to recognize and kill abnormal cells, especially tumour cells and virally infected ones, without previous activation. To recognize their targets, NK cells use a wide variety of surface receptors, both activating and inhibitory. If a ligand binds to an NK receptor, immune response is triggered. Examples of such ligand-receptor pairs are NKp80-AICL and NKRP1-LLT1 on human lymphocytes. Another ligand-receptor system of this kind is NKp65 and KACL, two recently discovered lectin receptors on human immunocytes. KACL is the last and most recently characterized member of CLEC2 receptor family in humans. Its expression is almost exclusively restricted to skin. NKp65, a close relative of NKp80, is a glycoprotein which stimulates NK92MI cell cytotoxicity upon KACL engagement. NKp65 has been shown to bind to KACL with a fairly high affinity by surface plasmon resonance measurement. This thesis aims at describing the cloning of expression vectors coding for NK cell receptors NKp65 and KACL, expression of these proteins in HEK293T cell line and their purification. Keywords: NKp65, KACL, NK cell, lectin, receptor, plasmid (in Czech)
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Molecular genetic characterization of vancomycin-resistant enterococci
Bubeníček, Karel ; Rada, Vojtěch (advisor) ; Igor, Igor (referee)
Summary
Objectives and hypothesis: This thesis concerns the study of plasmids of vancomycin-
resistant enterococci isolated from feces of American crows in the years 2012 - 2013 period.
The hypothesis is that, in various environments, there is one or more types of
epidemiologically significant vanA gene-carrying plasmids that are capable of horizontally
spread.
Methods: Based on PFGE method the number and size of plasmids were detected in selected
isolates of vancomycin-resistant E. faecium. Using PCR method the isolates were subjected
to detection of genes of replicases, relaxases and toxin-antitoxin system of plasmid-bound
resistance genes. Using 19 primers were characterized types of Tn1546.
Results: Of the 12 tested vancomycin-resistant isolates of E. faecium the following number
and size of plasmids was proven using PFGE method: 2 isolates contained two plasmids
(17%), 3 isolates contained three plasmids (25 %), 5 isolates contained four plasmids (42 %)
and 2 isolates contained five plasmids (17 %).
All isolates (n = 12) were then subjected to the detection of genes of replicases, relaxases and
toxin-antitoxin system for typing of plasmids from each plasmid families.
RepA_N family of plasmids:
genes characterizing plasmids related to pRUM: rep17 in 11 isolates (92 %),
gene Axe-Txe was detected in 5 isolates (42 %)
genes characterizing plasmids related to pLG1: rep20 in 7 isolates (58 %)
genes characterizing plasmids related to pAD1: relpAD1 gene was detected in one isolate (8 %)
Inc18 family of plasmids:
genes characterizing plasmids related to pIL501: rep1 gene detected in one case (8 %)
genes characterizing plasmids related to pRES25: rep2 gene in 2 isolates (17 %)
genes characterizing plasmids related to pEF1: relpEF1 detected in 11 isolates (92 %)
pHTB family of plasmids:
genes characterizing plasmids related to pHTB: rep22 gene was detected in 4 isolates (33%) and in 2 isolates gene relpHTB was detected (17%)
RCR family of plasmids:
genes characterizing plasmids related to pRI: positive detection of Rep14 gene in 8 isolates (67%) and in 4 isolates relpRI gene was detected
Small theta-replicating plasmids:
genes characterizing plasmids related to pEF418 plasmids: rep18a gene in 2 isolates (17%)
genes characterizing plasmids related to pB82: rep18b gene was detected in one isolate (8%)
genes characterizing plasmids related to pCIZ2: relpCIZ2 gene was detected in 9 isolates tested (75%)
Types of transposon Tn1546
Using the PCR method types of Tn1546 were characterized. In 4 isolates (n = 12; 33 %)
Tn1546 was characterized as a F3 type. In one isolate (8 %) Tn1546 was characterized as a
type F5, in one isolate (8 %) as a type PP-16. In 6 isolates Tn1546 was untypeable. Most
likely these are new, yet unknown types.
Conclusion: This is the first study of plasmids of vancomycin-resistant isolates E. faecium
isolated from feces of American crows. These results emphasize not only a high proportion of
plasmids in individual isolates, but also a high proportion of genes with horizontal transfer.
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Preparation and purification of plasmid DNAs
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of therapeutic methods in medicine, like are DNA vaccines and gene therapy increases demand for new processes for the isolation and purification highly pure plasmid DNA. Most often used methods of purification plasmid DNA are chromatographic methods. In experimental part of this thesis was performed isolation of plasmid pUC-19 DNA plasmid via alkalyne lysis. And purification of plasmid was performed by liquid chromatography and agarose gel electrophoresis.
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Plasmid DNA vaccines
Machan, Radoslav ; Chroboková, Maria (referee) ; Rittich, Bohuslav (advisor)
Plasmid DNA vaccines are the new generation of vaccines with a great potential in prevention of many diseases. Recent studies and clinical test are aimed at prevention against cancer, hepatitis, malaria, HIV, influenza and other diseases. Recent main challenges covering plasmid DNA vaccines are associated with optimalization of each step of production and mainly purification steps allowing production of pDNA at kilogram levels. Main purification techniques used are based upon chromatographic methods, but research and development also shows other potential methods, like two-phase aqueous systems or magnetic microparticles as carriers. In experimental part of this thesis isolation of pUC 19 plasmid from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Isolation was verified by agarose gel electrophoresis. Isolated samples were purified in four repetitions with lithium chloride and magnetic microparticle carriers and the extent of purification was verified spectrophotometrically. Purified samples were visualised via agarose gel electrophoresis and results were compared.
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