20.500.12556/RUL-172555 Načrtovanje in sestavljanje sintezne knjižnice nanoteles proti β-laktamazi BcII na osnovi ogrodja cAbBCII10 Design and construction of a synthetic nanobody library against β-lactamase BcII based on the cAbBCII10 nanobody framework Nanotelesa so enodomenski fragmenti kamelidnih težkoverižnih protiteles. Zaradi svoje velikosti imajo nanotelesa več prednosti pred klasičnimi protitelesi, zato jih uporabljamo na različnih področjih raziskovanja in medicine. Običajno nanotelesa pridobivamo z imunizacijo kamelidov, kar je zamuden in drag postopek. Obstaja tudi več alternativnih metod pridobivanja nanoteles brez imunizacije živali, med katerimi se v zadnjem času vedno bolj uveljavlja načrtovanje nanoteles z uporabo strojnega učenja. V magistrski nalogi smo načrtovali nova nanotelesa proti β-laktamazi BcII iz bakterije Bacillus cereus na osnovi ogrodja cAbBCII10. Za ta namen smo z orodjem AlphaFold 3 napovedali strukturo kompleksa cAbBCII10 in BcII, nato pa smo z metodami strojnega učenja napovedali nova nanotelesa, ki se vežejo na isti epitop kot cAbBCII10. Na podlagi zaporedij načrtovanih nanoteles smo pripravili knjižnico zaporedij, ki smo jo sestavili s cikličnim sestavljanjem s polimerazo. Nato smo z dvohibridnim sistemom kvasovke preverili vezavo načrtovanih nanoteles na BcII in preverili kvaliteto sestavljene knjižnice z metodami sekvenciranja naslednje generacije. Zaradi napak, ki so nastale pri sestavljanju knjižnice, nismo uspeli preveriti interakcije vseh načrtovanih nanoteles z BcII, kljub temu pa smo izolirali tri nova nanotelesa z nizko afiniteto do β-laktamaze BcII Nanobodies are single-domain fragments of camelid heavy chain-only antibodies. Due to their size, nanobodies possess several key advantages over classical antibodies and are used in research and medicine. Nanobodies are usually generated using camelid immunization which is time-consuming and expensive. Beyond animal immunization, multiple methods for identifying nanobodies have emerged with machine learning–guided design becoming increasingly popular. In this master's thesis we designed new nanobodies against BcII, a β-lactamase from Bacillus cereus, based on the cAbBCII10 scaffold. We used AlphaFold 3 to predict the structure of the complex between cAbBCII10 and BcII. Next, we used machine learning to design new nanobodies targeting the same epitope and constructed a DNA-library based on the designed nanobody sequences, which we assembled using polymerase chain assembly. Subsequently, we evaluated nanobody binding to BcII and used next generation sequencing to validate the quality of the constructed library. Due to mistakes that were introduced into the nanobody sequences during library assembly, we were not able to screen all the designed nanobodies for their ability to bind BcII. Despite this, we isolated three new nanobodies with low affinity for BcII. nanotelo načrtovanje proteinov dvohibridni sistem kvasovke nanobody protein design yeast two-hybrid true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-08 14:20:00 2025-09-08 14:20:06 2025-10-24 12:46:57 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25620 254673923 Krajnik_Bor_-_Nacrtovanje_in_sestavljanje_sintezne_knjiznice_nanoteles_proti_β-laktamazi_BcII_na.pdf Krajnik_Bor_-_Nacrtovanje_in_sestavljanje_sintezne_knjiznice_nanoteles_proti_β-laktamazi_BcII_na.pdf 1 CCA333F3607EC1E671913936473B3509 0a2c4dca38717d47d94115e1ce2e19f7a7640f7c0e841284b6b6873688a09316 02c9328a-8cae-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=216393 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0