20.500.12556/RUL-186877 Razvoj plazmidnega sistema in vrednotenje izražanja endonukleaze Cas9 v kvasovkah Hanseniaspora uvarum in Hanseniaspora opuntiae Development of a plasmid system and evaluation of Cas9 endonuclease expression in the yeasts Hanseniaspora uvarum and Hanseniaspora opuntiae V diplomski nalogi smo obravnavali vzpostavitev protokola za transformacijo nekonvencionalnih in slabo genetsko raziskanih vrst kvasovk Hanseniaspora uvarum in Hanseniaspora opuntiae. Obe vrsti sta zanimivi zaradi potencialne uporabe v mešanih fermentacijah z vrstami rodu Saccharomyces, kjer lahko prispevata predvsem k aromatični kompleksnosti vina. Za preverjanje postopka transformacije smo uporabili uveljavljen protokol za transformacijo Saccharomyces cerevisiae s plazmidom p414_HisCas9, medtem, ko smo za seva Hanseniaspora razvili prilagojen postopek transformacije z elektroporacijo. Za transformacijo smo uporabili plazmid pJJH3200_His_Cas9, ki ima mesto začetka pomnoževanja vrstno prilagojen. Transformante S. cerevisiae so uspešno rastle in po ponovitvi poskusa tudi tudi transformante H. uvarum, ko smo uporabili višjo začetno optično gostoto celic, medtem ko pri H. opuntiae transformant nismo dobili. Določanje celokupne koncentracije proteinov transformant H. uvarum je pokazalo večinoma negativne ali nizke vrednosti, zato prisotnosti in izražanja proteina Cas9 ni bilo mogoče zanesljivo potrditi. Rezultati kažejo, da je transformacija predstavnikov rodu Hanseniaspora metodološko zahtevna in da bodo za vzpostavitev zanesljivega sistema potrebne nadaljnje optimizacije transformacije, potrditve transformantov s PCR in izboljšave pristopov za detekcijo proteinov. In this B.Sc. thesis, we investigated the establishment of a transformation protocol for non-conventional and genetically poorly characterized yeast species Hanseniaspora uvarum and Hanseniaspora opuntiae. Both species are of interest due to their potential application in mixed fermentations with Saccharomyces species, where they can particularly contribute to the aromatic complexity of wine. To validate the transformation procedure, we used an established protocol for Saccharomyces cerevisiae transformation using the plasmid p414_HisCas9, whereas an adapted electroporation transformation procedure was developed for the Hanseniaspora strains. For the transformation, we used the plasmid pJJH3200_His_Cas9, which features a species-adapted origin of replication. S. cerevisiae transformants grew successfully, as did H. uvarum transformants after repeating the experiment with a higher initial optical density of cells, whereas no transformants were obtained for H. opuntiae. Determination of the total protein concentration of H. uvarum transformants mostly yielded negative or low values; therefore, the presence and expression of the Cas9 protein could not be reliably confirmed. The results indicate that the transformation of representatives of the genus Hanseniaspora is methodologically challenging and that further optimization of transformation, PCR validation of transformants, and improvements in protein detection approaches will be required to establish a reliable system. kvasovke Hanseniaspora uvarum Hanseniaspora opuntiae CRISPR-Cas9 plazmidni sistem transformacija kvasovk izražanje proteinov nekonvencionalne kvasovke yeasts Hanseniaspora uvarum Hanseniaspora opuntiae CRISPR-Cas9 plasmid system yeast transformation protein expression non-conventional yeasts true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-09-06 08:15:18 2026-09-06 08:15:22 2026-09-08 03:29:13 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 287174 290267651 61213.pdf 61213.pdf 1 D6E394D08D68E85392B4C6B32E9C1799 f5122b75a71255b3a1c5c7cf20b192b90e47269da3fd14b4a9255e55179f45e8 56d51d43-a9ba-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246259 Biotehniška fakulteta 0 0 0