20.500.12556/RUL-165879 Priprava in manipulacija umetnega kromosoma kvasovke Construction and manipulation of yeast artificial chromosome Umetni kromosomi kvasovke so bili v preteklosti sintetizirani z namenom vstavljanja velikih fragmentov za pripravo genomskih knjižnic. Ključno vlogo so imeli pri projektu Človeški genom, v zadnjih letih pa je zanimanje zanje v znanstveni sferi precej upadlo. Z napredkom molekularno-bioloških tehnik bi lahko bili ponovno zanimivi za uporabo kot platforma za vstavljanje, spreminjanje in izražanje rekombinantnih genetskih elementov v celicah kvasovk S. cerevisiae. V magistrskem delu smo pripravili posodobljen umetni kromosom kvasovke na osnovi vektorja pYAC4, kjer smo zamenjali telomerne regije, da je njegov skelet mogoče pomnožiti tudi z metodo PCR, in selekcijske označevalce za lažjo uporabo v laboratorijskih sevih kvasovke Saccharomyces cerevisiae. V tako pripravljen modificiran umetni kromosom kvasovke smo nato dodajali kasete z reporterskimi geni in primerjali učinkovitost sestavljanja skeleta in kaset med metodo sestavljanja po Gibsonu in metodo sestavljanja s homologno rekombinacijo v celicah kvasovke. Vstavljene kasete smo nato spreminjali in vivo z metodo CRISPR-Cas9 neposredno na umetnem kromosomu. Učinkovitost spreminjanja kaset smo ovrednotili na podlagi izražanja reporterskih genov ter preverjali, kakšen vpliv ima linearizacija umetnega kromosoma na sposobnost rasti transformant ter ali ima shranjevanje z zmrzovanjem negativen vpliv na ohranjanje umetnih kromosomov v kvasovkah. Ugotovili smo, da je sestavljanje s homologno rekombinacijo v kvasovkah S. cerevisiae časovno in delovno bolj obremenjujoče, daje pa boljše rezultate pri zlaganju več in kompleksnejših fragmentov, kot sestavljanje z metodo po Gibsonu. Kasete, vstavljene v umetni kromosom kvasovke, je mogoče spreminjati s sistemom CRISPR-Cas9 neposredno na kromosomu, linearizacija umetnih kromosomov in shranjevanje kvasovk pri -80 °C pa nimata negativnega vpliva na rast kvasovk. Yeast artificial chromosomes were originally synthesised to insert large fragments for the preparation of genomic libraries. They played a crucial role in the Human Genome Project, but in recent years the interest within the scientific community has decreased. Alas, with the advancements in molecular biology techniques, they could become a platform of interest for insertion, modification and expression of recombinant genetic elements in yeast cells. In this work, we prepared a modified yeast artificial chromosome based on the pYAC4 vector, where we replaced the telomeric regions to allow it to be amplified in multiple parts using the PCR method and replaced the selection markers for easier use in the laboratory strains of yeast Saccharomyces cerevisiae. We added cassettes to this modified yeast artificial chromosome for phenotypic monitoring of the inserts and compared the efficiency of the backbone and cassette assembly between the Gibson assembly method and homologous recombination in the yeast cells. The inserted cassettes were then modified in vivo using the CRISPR-Cas9 method directly on the artificial chromosome. The efficiency of cassette modification was evaluated phenotypically, we examined the impact of chromosome linearization on the growth of transformants and whether storage including freezing negatively affects the preservation of artificial chromosomes in yeast. We found out that homologous recombination in yeast cells is more time-consuming and labor-intensive than Gibson assembly but yields better results when assembling multiple and more complex fragments. The cassettes in the yeast artificial chromosome can be modified using the CRISPR-Cas9 system directly on the chromosome, and linearization of artificial chromosomes and storage of yeast cells at -80 °C do not have a negative impact on the yeast cell growth. umetni kromosom kvasovke pYAC4 Saccharomyces cerevisiae CRISPR-Cas homologna rekombinacija sestavljanje po Gibsonu EGFP EBFP LacZ KanMX yeast artificial chromosome pYAC4 Saccharomyces cerevisiae CRISPR-Cas homologous recombination Gibson assembly EGFP EBFP LacZ KanMX true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-12-12 14:18:29 2024-12-12 14:18:39 2024-12-16 14:48:47 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 255828 219310339 45442.pdf 45442.pdf 1 CB613965DAC4B9489038660E5E18E8F5 ff46e6116fe5caed96e55e1e613047a5454dbaf2b34d445dc5fd029db5940932 1a2bebdd-b88b-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=197148 Biotehniška fakulteta 0 0 0