20.500.12556/RUL-103961 Inaktivacija genskih skupin izbranih biološko aktivnih sekundarnih metabolitov v bakterijah vrste Streptomyces rapamycinicus. Inactivation of gene clusters for chosen bioactive secondary metabolites in bacteria Streptomyces rapamycinicus Sekundarni metabolizem streptomicet je pogosto izredno kompleksen in raznovrsten. Njihovi genomi imajo zapis za biosintezo številnih biološko aktivnih učinkovin, med katerimi številne izkazujejo protibakterijsko delovanje. Napredek na področju identificiranja genskih gruč in njihove anotacije v genom organizmov iz rodu Streptomyces, zahteva razvoj novih metod genske manipulacije. Metode genske manipulacije in ovrednotenje biosinteznih poti predstavljajo enega izmed pristopov pri opredelitvi novih biološko aktivnih učinkovin z zanimivim delovanjem. V sklopu magistrskega dela smo preučevali organizem S. rapamycinicus, znanega producenta rapamicina. Namen našega dela je bil ovrednotiti različne pristope za ciljno inaktivacijo genov znanih genskih skupin (rapamicinska, nigericinska in elaiofilinska) pri izbranem organizmu. Z uporabo molekularno bioloških metod smo načrtovali orodja na osnovi CRISPR-Cas9 metode preurejanja DNA in pripravili plazmide za homologno rekombinacijo. Uspeli smo pridobiti mutante, ki imajo prekinjene odseke v genskih gručah za biosintezo rapamicina, elaiofilina ali nigericina. V nadaljevanju smo pridobili še sev, ki ima prekinjeno tako biosintezo rapamicina kot tudi elaiofilina. Poleg tega smo v S. rapamycinicus pokazali funkcionalnost delovanja poročevalskega sistema na osnovi gena gusA, kot orodja za učinkovitejšo izvedbo genskega inženiringa. Secondary metabolism in Streptomyces species is often extremely complex and heterogeneous. These bacteria can biosynthesize many bioactive compounds, of which many display antibacterial activity. Rapid identification of gene cluster and their annotation in Streptomyces genomes requires development of novel methods for gene manipulation. Gene manipulation methods and characterisation of biosynthetic pathways is on of approaches which can be applied for identification of novel bioactive compounds with interesting activities. Our work was carried out on S. rapamycinicus, known as producer of rapamycin, and we aimed to evaluate different approaches in gene disruption of known gene clusters present in S. rapamycinicus, such as rapamycin, elaiophylin, nigericin. Methods of molecular biology were used to design CRISPR-Cas based tools for DNA rearangement and plasmids for homologous recombination were constructed. We completed succesful deletion of the parts of gene clusters encoding rapamycin, elaiophylin or nigericin. Double rapamycin-elaiophylin knock-outs were also completed. During our work we also managed to prove funcionality of gusA reporter system in S. rapamycinicus as a tool for efficient genetic engineering. streptomicete Streptomyces rapamycinicus genetski inženiring inaktivacija genov sekundarni metaboliti Streptomyces Streptomyces rapamycinicus genetic engineering gene inactivaciton secondary metabolites true false false [D. Vogrinc] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2018-09-29 07:45:40 2018-09-29 07:45:41 2022-08-17 03:42:25 0000-00-00 00:00:00 2018 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.3:579.873.7:604.4:615.281.9(043.2) 159179 9053305 4293.pdf 4293.pdf 1 A65F209DFC2C1A6C7C7F31247659995C 208c70e8d119c34b13a3ce1e1ec3d5d3c5590eaf89776fc4edb630775e4617c7 f2a98079-a1b5-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=114470 Biotehniška fakulteta 0 0 0