20.500.12556/RUL-148048 Poskus inženiringa reporterskega faga za detekcijo bakterije Staphylococcus aureus An attempt to engineer a reporter phage for the detection of Staphylococcus aureus Pomanjkanje učinkovite metode za detekcijo bakterije Staphylococcus aureus je velik izziv za javno zdravstvo, saj je le-ta odgovorna za visoke stopnje obolevnosti in smrtnosti po vsem svetu. V magistrskem delu smo želeli predstaviti inovativno strategijo, ki združuje prednosti fagnih delcev in proteina GFP, za hitro in učinkovito detekcijo bakterije S. aureus. Z vključitvijo gena gfp v fagni genom, se lahko gen gfp med okužbo tarčne bakterije izrazi in omogoči zaznavanje produkta z merjenjem fluorescence. Reporterski fag smo želeli konstruirati z uporabo sistema CRISPR-Cas9, ki bi v fagni genom uvedel dvojni zlom in posredoval rekombinacijo med fagnim genomom in donorsko DNK. Za to je potrebna elektroporacija celic S. aureus s plazmidom z zapisom za gen gfp, obdan s homolognimi regijami, in komponentami sistema CRISPR-Cas9. V predstavljenem delu smo preizkusili različne pogoje elektroporacije celic S. aureus, vendar nam s pripravljenimi elektrokompetentnimi celicami ni uspelo pridobiti transformant. Nazadnje smo uporabili komercialne elektrokompetentne celice in uspeli pridobiti dve transformanti, od katerih smo v eni potrdili prisotnost vstavljenega plazmida z metodo PCRKOL. Ob izolaciji plazmidne DNK iz transformiranih celic smo dobili zelo nizke donose, kar je lahko posledica slabe lize celic, majhnega števila kopij plazmida, uporabe celic v neustrezni fazi bakterijske rasti in nestabilnosti ali toksičnosti plazmida. Zaradi časovnih omejitev v okviru magistrske naloge, nadaljnjih korakov načrtovanega poskusa nismo izpeljali. Lack of an effective method for detection of Staphylococcus aureus is a major challenge for public health, as the bacterium is responsible for high rates of morbidity and mortality worldwide. In this thesis, we aimed to introduce an innovative strategy that combines the advantages of phage particles and the GFP protein for a rapid and efficient detection of S. aureus. By incorporating the gfp gene into the phage genome, GFP can be expressed during infection of the target bacteria, enabling detection by measuring fluorescence. We attempted to construct a reporter phage using the CRISPR-Cas9 system, which would introduce a double-strand break in the phage genome and mediate recombination between the phage genome and donor DNA. This requires electroporation of S. aureus with a plasmid containing the gfp gene, flanked by homologous regions and components of the CRISPR-Cas9 system. We tested various conditions for electroporation of S. aureus, but haven't been able to obtain transformants with electrocompetent cells prepared by standard protocol. Finally, we used commercial electrocompetent cells and managed to obtain two transformant colonies. In one of the transformants, the presence of the insert within the plasmid was also confirmed by the PCRKOL method. However, we obtained very low yields of isolated plasmid DNA from the transformed cells. This may be associated with poor cell lysis, low plasmid copy number, harvesting cells at an inappropriate stage of bacterial growth, plasmid instability or plasmid toxicity. Due to time constraints within the scope of the master's thesis, we were unable to proceed with further planned steps of the experiment. Staphylococcus aureus elektroporacija protein GFP reporterski fag urejanje fagnega genoma CRISPR-Cas9 Staphylococcus aureus electroporation GFP protein reporter phage phage genome editing CRISPR-Cas9 true false false [K. Malalan] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-07-26 12:48:58 2023-07-26 12:49:08 2023-07-27 09:54:51 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.621:579.86 221656 159865603 33120.pdf 33120.pdf 1 4E2E48B8E23767802749BB96DBB387E2 2f0173b810594c8b124eb39805a3cf4fcafd4f3373e00f23d04cbdbce4d7bcc0 024a8b0c-2ba2-11ee-aeaf-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=171982 Biotehniška fakulteta 0 0 0