20.500.12556/RUL-181009 Vrednotenje izločanja heterolognih proteinov iz bakterije Streptomyces rimosus s prilagoditvijo presejalnega testa na manjše merilo Evaluation of heterologous proteins secretion from Streptomyces rimosus using scaled-down screening procedure Bakterije rodu Streptomyces so znane po proizvodnji številnih sekundarnih metabolitov, kot je na primer široko spektralni antibiotik oksitetraciklin. V preteklem desetletju pa so aktinomicete vzbudile zanimanje raziskovalcev, kot celične tovarne za izražanje heterolognih proteinov. V zasnovi te magistrske naloge smo izbrali tri seve Streptomyces rimosus, zasnovane za izločanje proteaze kaminizina, s poročevalskim sistemom fluorescentnega proteina mCherry. V eksperimentalnem delu smo z namenom optimizacije standardnega biosinteznega procesa opredelili morfologijo rasti izbranih sevov v tekočem gojišču, testirali vpliv različnih vrst inokuluma na rast kulture in izločanje heterolognega proteina. Optimizacijo procesa smo primerjali v volumnih 40 mL in 5mL in izvedli prehod na manjšo skalo v 3 mL. Ob uspešnem prehodu na manjše merilo smo s ciljem po povečanem izločanju proteina mCherry raziskali možnosti re-optimizacije GOTC gojišča. Zamenjava vira dušika iz kazeinskega triptona na sojine beljakovinske koncentrate in sojine moko je privedla do znatne spremembe v izločanju heterolognega proteina kaminizina. Po zamenjavi triptona, smo z uporabo beljakovinskega koncentrata iz sojine moke dosegli vsaj 30 % višje vrednosti fluorescence poročevalskega sistema mCherry. Members of the Streptomyces genus are known for the production of various secondary metabolites, such as broad spectrum antibiotic oxytetracycline. In the past decade, however, actinomycetes have raised the interest of researchers as candidate cell factory for heterologous protein expression. In the scope of this work, we selected three Streptomyces rimosus strains, constructed and tested for the expression of heterologous gene encoding caminisine, fused with the fluorescent protein mCherry reporter system. In the experimental part, with the aim of re-optimizing the standard bioprocess, we defined the growth morphology of the selected strains in liquid media, tested the influence of various inoculum sources on culture growth and protein production. Optimization was carried out at 40 mL and 5 mL scale, which was scaled down to a smaller scale of 3 mL. Upon successful transition to a smaller scale, we explored the possibilities of modifying the standard growth medium to accomodate higher bioprocess productivity. Replacing the nitrogen source casein tryptone by soy flour and soy protein concentrate led to a significant change in the production of the heterologous protein. Compared to tryptone, we achieved at least 30 % higher fluorescence values of the mCherry in the medium containing soy flour protein isolate. streptomicete Streptomyces rimosus heterologno izražanje kaminizin mCherry presejalni test fluorescenca vir dušika optimizacija gojišč Streptomyces Streptomyces rimosus heterologous expression caminisine mCherry screening fluorescence nitrogen source growth medium optimization true false false [G. Medved] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-03-22 07:15:52 2026-03-22 07:15:59 2026-03-23 09:00:01 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.3/.4:579.873.7 279195 272567043 56980.pdf 56980.pdf 1 01E7CDA423DCC6D288C944A432CF6CBF fdef9a088b4e7af488a7f46d5eed980f65a52db4836d2d4e9d7c75ff6c39d37b 7f7b18c1-25b6-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=231062 Biotehniška fakulteta 0 0 0