20.500.12556/RUL-127209 Delecija genske skupine za biosintezo oksitetraciklina v bakteriji Streptomyces rimosus z metodo homologne rekombinacije Deletion of oxytetracycline gene cluster in Streptomyces rimosus with homologous recombination Bakterija Streptomyces rimosus je ena izmed najbolje preučenih industrijskih streptomicet, ki proizvaja antibiotik oksitetraciklin (OTC). Zaradi visokega donosa OTC je sev S. rimosus atraktiven gostitelj za heterologno produkcijo naravnih učinkovin poliketidnega izvora. Namen te magistrske naloge je bil pridobiti sev S. rimosus z delecijo genske gruče za biosintezo oksitetraciklina. Delecijo smo izvedli z metodo homologne rekombinacije. Konstruirali smo temperaturno občutljiv plazmid, ki vsebuje vse elemente sistema homologne rekombinacije. Uporabili smo homologne sekvence različnih dolžin, ki so nalegale na začetek in konec otc genske gruče. Kolonije z ustrezno delecijo smo iskali z metodo subkultivacij in s precepljanjem kolonij na agarske plošče z in brez dodanega selekcijskega markerja (eritromicin). Pri pridobljenih mutantah, ki niso proizvajale OTC, smo delecijo potrdili na genetski ravni s PCR in s sekvenciranjem ter na metabolni ravni s tankoplastno kromatografijo (TLC) in s tekočinsko kromatografijo (LC). Streptomyces rimosus produces antibiotic oxytetracycline (OTC) and is one of the most intensively studied streptomyces species. High yields of oxytetracycline makes S. rimosus an attractive host for potential heterologous production of polyketide based secondary metabolites. The objective of this thesis was to obtain an S. rimosus strain with the deletion of otc gene cluster. The deletion was carried out with the method of homologous recombination. In order to complete deletion, we constructed a temperature sensitive plasmid vector with all the necessary elements to carry out homologous recombination experiment. Homologous sequences of different lengths were designed to anneal on both sides of otc gene cluster. To obtain potential mutants we used a method of subcultivation and patching on agar plates with and without added selection marker (erythromycin). Colonies which did not show any OTC production were tested with polymerase chain reaction (PCR), sequencing, thin layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). aktinomicete Streptomyces rimosus tetraciklini delecija GBG homologna rekombinacija actinomycetes Streptomyces rimosus tetracyclines gene cluster deletion homologous recombination true false false [M. Rebek] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-05-26 07:15:10 2021-05-26 07:15:22 2022-09-06 03:50:09 0000-00-00 00:00:00 2021 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2023-05-20 602.3:579.873.7:602.6:579.8:604.4:615.33:606:62(043.2) 191210 65331971 13959.pdf 13959.pdf 1 4026B5ED509EEA7C0699E2530BDAC688 1d1ee66b7cffc80bb0ffe5bb3836138e4adc7bcf9fcd538a65485ac15334d540 5cee4321-bde1-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=143493 Biotehniška fakulteta 0 0 0