20.500.12556/RUL-177304 Transformacija bakterij Pseudomonas spp. s plazmidom z zapisom za fluorescentni in/ali luminiscentni protein Transformation of Pseudomonas spp. with a fluorescence and/or luminescence protein encoding plasmid Bakterije vrste Pseudomonas fragi in Pseudomonas aeruginosa zaradi sposobnosti tvorbe biofilma pomenijo velik izziv, saj jim biofilm omogoča preživetje tudi v neugodnih pogojih zdravstvenega in industrijskega okolja. Biofilmi bakterijam zagotavljajo dodatno zaščito pred zunanjimi vplivi, kar otežuje njihovo odstranjevanje. V magistrski nalogi smo vzpostavili metodo za detekcijo in kvantifikacijo bakterij P. fragi z uporabo fluorescentnih proteinov. Najprej smo pripravili plazmida pBBR1MCS-2_mTagBFP2 in pBBR1MCS-2_mCherry, ki nosita gene za izražanje fluorescentnih proteinov mTagBFP2 in mCherry, ter z elektroporacijo izvedli transformacijo bakterij. Pridobljene transformirane bakterije P. fragi in P. aeruginosa, ki so izražale fluorescentne proteine, smo uporabili za identifikacijo bakterij v planktonskih obliki in v obliki biofilmov. Meritve intenzitete flourescentne svetlobe so pokazale korelacijo med rastjo bakterij in intenziteto izražanja fluorescentnih označevalcev pri bakteriji P. fragi, s čimer smo potrdili zanesljivost predlagane metode. Vzpostavljena metoda je hitra, natančna in ponovljiva, kar omogoča natančno kvantifikacijo bakterijskih populacij v različnih eksperimentalnih pogojih. Poleg tega so transformirane bakterije P. fragi ohranile sposobnost tvorbe biofilmov, kar potrjuje uporabnost fluorescentnih označevalcev za preučevanje rasti bakterij in njihove pritrditve na površine. Vzpostavljena metoda je nov način detekcije bakterij v obliki biofilma, kar odpira nove možnosti za razvoj strategij za preprečevanje in nadzor biofilmov ter zmanjšanje tveganj za bakterijske kontaminacije in okužbe. The bacteria Pseudomonas fragi and Pseudomonas aeruginosa pose a major challenge due to their ability to form biofilms, which enable them to survive even in the unfavorable conditions of healthcare and industrial environments. Biofilms provide bacteria with additional protection against external influences, making them difficult to remove. In master's thesis, we established a method for the detection and quantification of P. fragi bacteria using fluorescent proteins. First, we prepared plasmids pBBR1MCS-2_mTagBFP2 and pBBR1MCS-2_mCherry, which carry genes for the expression of fluorescent proteins mTagBFP2 and mCherry, and performed bacterial transformation using electroporation. The obtained transformed P. fragi and P. aeruginosa bacteria, which expressed fluorescent proteins, were used to identify bacteria in planktonic form and in the form of biofilms. Measurements of fluorescent light intensity showed a correlation between bacterial growth and the intensity of fluorescent marker expression in P. fragi bacteria, confirming the reliability of the proposed method. The established method is fast, accurate, and reproducible, enabling accurate quantification of bacterial populations under various experimental conditions. In addition, the transformed P. fragi bacteria retained their ability to form biofilms, confirming the usefulness of fluorescent markers for studying bacterial growth and their attachment to surfaces. The established method represents a new way of detecting bacteria in the form of biofilms, opening up new possibilities for the development of strategies for the prevention and control of biofilms and the reduction of the risks of bacterial contamination and infection. Pseudomonas fragi Pseudomonas aeruginosa fluorescenca mTagBFP mCherry planktonske celice biofilm Pseudomonas fragi Pseudomonas aeruginosa fluorescence mTagBFP mCherry planktonic cells biofilm true false false [M. Dostal] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-12-20 07:15:25 2025-12-20 07:15:39 2025-12-30 03:44:14 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 579.254.2:579.841.1 277594 262478083 55600.pdf 55600.pdf 1 482878BA5D4F55E7F87F2D1DB5F85750 8915536c58aca2c90637d6ff7a2dc34e9b761f07265d2a4e2888759dac18954b eba2dd81-dd6a-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=224267 Biotehniška fakulteta 0 0 0