Original title:
Hybridní faktory sigma RNA polymerasy u Corynebacterium glutamicum
Translated title:
Hybrid sigma factors of RNA polymerase in Corynebacterium glutamicum
Authors:
Blumenstein, Jan ; Štěpánek, Václav (advisor) ; Krásný, Libor (referee) Document type: Master’s theses
Year:
2019
Language:
cze Abstract:
[cze][eng] půdní bakterie využívaná průmyslu jako producent aminokyselin, nukleotidů, biopaliv a alkoholů. Cílem této práce bylo vytvořit hybridní faktor σ rozpoznávat pouze příslušný hybridní promotor, takže neovlivní e genů. Na základě aminokyselinových sekvencí faktorů σ a σ genů navrženy 2 typy hybridních faktorů σ σ σ σ byly dále na základě přirozeného faktoru σ σ promotoru. Hybridní promotory byly vytvořeny kombinací 10, které pocházely ze σ a σ ů promotorů s použitím prokázalo, že exprese genu σ σ kmeny, které nesly přirozený faktor σ a příslušný promotor. Při použití jednoho z ů σ σ ) se záměnami 35 σ promotorů došlo k a tedy dosažení cíle. Klíčová slova:Corynebacterium glutamicum is a Gram-positive non-sporulating soil bacterium which is used in biotechnology as a producer of amino acids, nucleotides, biofuels and alcohols. The aim of this thesis was to create a hybrid σ factor of RNA polymerase which would be able to recognize a matching hybrid promoter without effect on expression of the host genes. Based on the σD and σH amino acid sequence, two types of hybrid factors, σDH and σHD , were designed by the sequence combination of sigD and sigH. As an alternative approach, based on the in silico homology modeling, mutations of wild-type σH in the region recognizing the -35 promoter element of the σH -dependent promoter were introduced. Hybrid promoters were constructed by combining the -35 and -10 promoter regions that were derived from the σD - and σH - dependent promoters. Promoter activity was determined by using gfpuv reporter gene under the control of hybrid promoter. The expression of gfpuv in strains with hybrid sigma factors σDH / σHD and hybrid promoters was rather low compared to strains that carried wild-type σ factor and the respective promoter. The aim of the thesis was achieved by using one of the mutant σH factor (σmutH_6A ) with alterations in the region recognizing the -35 element of the σH -dependent promoter. This mutant σ...
Keywords:
Corynebacterium glutamicum; promoter; sigma factor; Corynebacterium glutamicum; faktor sigma; promotor
Institution: Charles University Faculties (theses)
(web)
Document availability information: Available in the Charles University Digital Repository. Original record: http://hdl.handle.net/20.500.11956/109533