20.500.12556/RUL-110585 Zaznavanje majhnih molekul z uporabo ločenih verig protiteles Small molecule sensing using separate variable antibody fragments Sintezna biologija stremi k pripravi novih molekularnih metod za preučevanje in uravnavanje procesov v celicah sesalcev. Diplomska naloga predstavlja nov, z majhnimi molekulami nadzorovan dimerizacijski sistem na osnovi ločenih enoverižnih fragmentov protiteles. Za dokaz koncepta smo si izbrali enoverižni variabilni fragment protitelesa proti estradiolu v fuziji s kresničkino luciferazo, ki je delovala kot poročevalski sistem. Z dodatkom estradiola v gojišče smo v celicah sesalcev uspešno sprožili dimerizacijo ločeno izražene lahke in težke verige protitelesa. Z MTT testom smo dokazali, da sistem deluje tudi pri koncentraciji estradiola, ki za celice ni toksična. S takšnim sistemom bi lahko na preprost način nadzorovali celične procese, kot so transkripcija, lokalizacija proteinov ali celična signalizacija. V delu smo predstavili le eno izmed protiteles in ligandov, sistem pa bi z uporabo drugih protiteles in ligandov lahko postal univerzalen. Synthetic biology strives to engineer new molecular methods for investigation and regulation of mammalian cell processes. This thesis presents a new dimerization system, controlled by small molecules, based on separated single chain Fab variable fragments. To prove the concept, we chose the anti-β-estradiol single chain Fab variable fragment, fused to a firefly’s luciferase, which served as the reporter system. By adding estradiol in the media of mammalian cells, we successfully initiated the dimerization of the individually expressed light- and heavy- antibody chains. The MTT assay proved that the system functions even when the estradiol concentration is not toxic to cells. With this system, we could easily control cell processes such as transcription, protein localisation and cellular signallization. While we examined only one of the antibodies and ligands, the application of others can make this system universal. Sintezna biologija kemijsko inducirana dimerizacija cepljena protitelesa scFv Synthetic biology chemically induced dimerization split antibody scFv true false false [Ž. Kolenc] Slovenski jezik Angleški jezik Diplomsko delo/naloga 2019-09-18 07:45:03 2019-09-18 07:45:12 2022-08-22 03:44:40 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2022-09-12 577.2(043.2) 169113 9364601 7994.pdf 7994.pdf 1 8D33BB840195BB0CEFC481C41B1385EC bcc016b00c4ede42b32c0fa97faebfa929d2aa287c4d694a65a1a600f2a52fdb 26d9af98-a1b7-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=122259 Biotehniška fakulteta 0 0 0