20.500.12556/RUL-152758 Spremljanje izražanja genov s fluorescenčnimi poročevalci v realnem času pri bakterijah Streptomyces spp. Real-time monitoring of gene expression based on a fluorescence reporter in Streptomyces spp. Zaradi potrebe po neinvazivni metodi za spremljanje fluorescence v realnem času v rastočih bakterijskih kulturah smo skupaj s podjetjem PreSens razvili merilec mSDR. Inovativno orodje, ki omogoča sočasno spremljanje rdeče fluorescence v do 240 vzorcih. Orodje smo vzpostavili, kalibrirali, preverili zmogljivost in območje detekcije signala. Njegovo uporabno vrednost smo preverili na primeru bakterije Streptomyces clavuligerus in poročevalskega sistema za aktivnost regulatorja CcaR, pozitivnega regulatorja gruče genov za biosintezo klavulanske kisline. Poročevalski sistem je temeljil na promotorju PceaS2, ki je vezavno mesto aktivatorja in promotorska regija prvega gena v gruči za biosintezo klavulanske kisline, in poročevalskem proteinu mCherry. Sklepali smo, da bo povečana aktivnost regulatorja CcaR vodila v močnejšo transkripcijo tako poročevalskega gena kot genov gruče klavulanske kisline. S pomočjo poročevalskega sistema in orodja mSDR smo natančneje spoznali mehanizme regulacije CcaR in izražanja genov za biosintezo klavulanske kisline. Ugotovili smo, da lahko na podlagi spremljanja fluorescence prepoznamo seve z boljšo produktivnostjo in večjim izkoristkom. Odkrili smo nove potencialne pristope za izboljšanje produkcijskih sevov in izrazito izboljšali produktivnost izbranega seva. Zasnovano orodje se je izkazalo kot izredno uporabno za spremljanje aktivnosti in regulacije izražanja genov vključenih v sintezo pomembnih sekundarnih metabolitov ter za izbor potencialnih sevov na laboratorijskem nivoju. Še več, orodje ima potencial za uporabo za različne analitske namene v mikrobiologiji in širše. To address the lack of analytical tools for non-invasive, on-line fluorescence determination in growing bacterial cultures, we collaborated with PreSens to develop mSDR, an innovative tool for simultaneous red fluorescence determination in up to 240 growing cultures. We developed, established, and calibrated the system, evaluated its precision and signal detection range. To assess the utility value of the mSDR and investigate the regulation of early clavulanic acid biosynthesis genes in Streptomyces clavuligerus, we designed reporter systems. These systems utilized the mCherry fluorescent protein under the control of the PceaS2 promoter. This promoter corresponds to the region of the ceaS2 gene, which is the first among the early genes in the clavulanic acid biosynthesis cluster. It is well-established that initiating transcription of the ceaS2 gene triggers the transcription of other genes in the cluster. The primary activator for this cluster is CcaR, and its binding site resides on the PceaS2 promoter. Therefore, we posited that increased CcaR regulator activity would lead to enhanced transcription of both the reporter gene and the clavulanic acid cluster genes. Through the monitoring of fluorescence, we can now identify bacterial strains with improved productivity and efficiency. We discovered new potential approaches to enhance production strains and significantly improved productivity of the selected strain. mSDR has proven to be extremely useful for monitoring the activity and regulation of genes involved in important secondary metabolites, as well as selecting potential production strains at the laboratory level. Furthermore, the tool has the potential for various analytical purposes in microbiology and beyond. neinvazivne analitske metode poročevalski sistem klavulanska kislina Streptomyces clavuligerus regulacija izražanja genov non-invasive analytical methods reporter system clavulanic acid Streptomyces clavuligerus gene expression regulation true false false [I. Fazarinc] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-12-06 10:32:21 2023-12-06 10:32:34 2023-12-15 12:04:23 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.3:579.873.7:575.117 236051 177664003 38215.pdf 38215.pdf 1 85FB9E98F90C7542A244F670C1875FC4 0ce711d7d66316dcee292d8cc511e0666d18926a5ce209a7a25984026e903337 640868ab-941a-11ee-ac6f-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=178392 Biotehniška fakulteta 0 0 0