20.500.12556/RUL-130412 Izbris daljšega genomskega odseka bakterije Streptomyces rimosus z uporabo CRISPR-Cas9 Large genomic deletion in bacteria Streptomyces rimosus using CRISPR-Cas9 Razvoj celičnih tovarn na osnovi bakterij iz rodu Streptomyces je ključen za proizvodnjo novih in že poznanih biološko aktivnih učinkovin. S postopno delecijo večjih delov genoma lahko razvijemo učinkovito platformo za heterologno izražanje sekundarnih metabolitov. Klasične metode za manipulacije genoma v streptomicetah so zahtevne in časovno potratne. V tem delu, smo z uporabo CRISPR-Cas9 sistema izvedli delecijo genoma bakterije Streptomyces rimosus v velikosti 145 kb. Z bioinformacijsko analizo smo izbrali tarčno regijo genoma, ustrezne homologne regije in vodilne RNA. Z metodama PCR s prekrivanjem in SLiCE kloniranje smo sestavili plazmidni konstrukt za delecijo s CRISPR-Cas9. Konstrukt smo s konjugacijo vnesli v S. rimosus in testirali delecijo v pozitivnih klonih z metodo PCR in sekvenciranjem. Identificirali smo številne seve s pravilno delecijo izbranega fragmenta DNK v velikosti 145 kb. Developing a Streptomyces-based cell factory is key for the production of new or known biologically active compounds. With gradual deletion of large genomic regions one can develop an efficient platform for the heterologous expression of secondary metabolites. Gene manipulations of Streptomyces using classical approaches are demanding and time-consuming. In this work we used CRISPR-Cas9 to achieve a 145 kb genomic deletion in Streptomyces rimosus. With the use of bioinformatics we chose a target region with accompaning homologies and guide RNAs. We then used overlap PCR and SLiCE cloning to construct a plazmid for CRISPR-Cas9 driven deletion. The plasmid was introduced into S. rimosus with conjugation, then positive clones were tested for the deletion using PCR and sequencing. We have identified several isolates with correctly deleted DNA fragment of 145 kb in size. CRISPR delecija redukcija genoma Streptomyces rimosus CRISPR genome reduction genomic deletion Streptomyces rimosus true false false [T. Godec] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-09-15 07:15:34 2021-09-15 07:15:37 2023-09-20 14:01:00 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 2024-09-09 602.3:579.873.7:602.6:579.8(043.2) 196299 76574723 19405.pdf 19405.pdf 1 7D59CEA9FE94F5C00A27C62E3C3BDD18 ba1f6ae471343cb60776773be8894e78128378c9c2fbc1b50d6a2450a48a4dd6 b52d67b4-15e3-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=147519 Biotehniška fakulteta 0 0 0