doktorska disertacija
Tina Fink (Author), Roman Jerala (Mentor)

Abstract

S sintezno biologijo in implementacijo logičnih vezij v žive organizme se je začelo tako imenovano biološko računalništvo. Sprva v bakterijah, kjer so že leta 2000 v E. coli pripravili prva kompleksna sintetična logična vezja na osnovi regulacije prepisovanja genov. Do danes so pripravili že več različnih celičnih vezij tako v bakterijah, kvasovkah kot tudi v sesalskih celicah. Večinoma vsa vezja temeljijo na uravnavanju prepisovanja genov, kar je sicer lažje načrtovati, vendar je odzivni čas takšnih celičnih vezij relativno počasen in ni primeren za aplikacije, ki zahtevajo odziv na spremembe v okolju v nekaj minutah. Zato ni presenetljivo, da je velik izziv sintezne biologije predstavljalo prav povečanje hitrosti procesiranja informacij v celicah. Signalno pot smo zasnovali na proteinskih interakcijah z obvitimi vijačnicami ter modifikacijah proteinov s proteolizo in se s tem izognili dolgotrajnejšim procesom prepisovanja in prevajanja genov. Pripravili smo nabor ortogonalnih razcepljenih proteaz, katerih aktivnost smo nadzorovali z zunanjimi signali, ter nabor obvitih vijačnic z avtoinhibitorno domeno. Z uvedbo cepitvenih mest za proteaze v zanki med antiparalelnima dimeroma ter v povezovalnem segmentu med segmentom obvite vijačnice ter funkcionalno domeno je dimerizacija obvitih vijačnic postala odvisna od proteolize, kar nam je omogočilo izvedbo vseh dvovhodnih Boolovih logičnih operacij v sesalskih celicah. V sesalskih celicah smo pokazali, da se celice s takšno signalno potjo lahko na zunanje signale odzovejo že po 15 minutah, kar je bistveno hitreje od tega kar zmorejo dosedanji sistemi. S primerjavo uravnavanja prepisovanja genov in aktivacije s proteolizo smo pokazali, da signalna pot na osnovi uravnavanja prepisovanja genov za primerljiv odziv potrebuje vsaj 2 uri. Poleg hitrosti je pomembna lastnost tega sistema tudi možnost združevanja reakcij v vzporedne in verižne reakcije ter kaskade. Z izvedbo proteaznega inverterja ter tristopenjske proteazne kaskade smo pokazali tudi modularnost in razširljivost signalne poti. Z uvedbo dodatnih proteaz, kot je proteaza HIV-1, ter s prenosom signalne poti v druge celične linije smo pokazali, da je zasnovana signalna pot robustna in neodvisna od endogenih celičnih procesov.

Keywords

logična vezja;biološko računalništvo;signalne poti;razcepljene ortogonalne proteaze;sesalske celice;biološki sistemi;uravnavanje;biokemija;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: [T. Fink]
UDC: 004.9:577.25(043.3)
COBISS: 301627904 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Split orthogonal protease-based logic circuits
Secondary abstract: Synthetic biology introduced the implementation of logic circuits into living organisms, thus triggering the onset of biological computing. Complex logic circuits, based on transcriptional regulation, were first assembled in E. coli already in 2000. Since then, various cellular circuits were established in bacteria, yeast as well as in mammalian cell lines. Due to ease of design, most of the approached employ transcriptional regulation as the underlying mechanism. Due to relatively slow response time, these circuits are not particularly suitable for applications, which demand response of the system in minutes. It is not surprising that the information processing time in the cells has become one of the big challenges of synthetic biology. Therefore, we have rather based a signalling pathway on protein interactions with coiled-coils and proteolytic cleavage, thus avoiding the lengthy processes of transcription and translation in response to a signal. We have designed a set of orthogonal split proteases, combined with a set of coiled-coils with autoinhibitory domains. By introducing protease cleavage sites into loops between the antiparallel coiled-coil dimers, and into the linker connecting the coiled-coil segment with the catalytic domain, we have made the dimerization of the coiled-coils dependent of proteolysis. This allowed design of all Boolean logic circuits in mammalian cells, which respond to external signals. In mammalian cells we have achieved response times of 15 minutes, which is significantly faster compared to existing approaches. Compared to our proteolytic design, we have shown that the signalling pathways relying on transcriptional regulation only reach comparable response in 2 hours. In addition to a fast response, our system allows cascading of the individual logical operations in to complex parallel or serial systems. By introduction of a protease inverter and a three-step proteolytic cascade we have demonstrated modularity and scalability of the system. In addition, using additional proteases, such as HIV-1 protease, and transfer of the system into distinct cell lines we have shown that the system is robust and independent of the intrinsic cellular processes.
Secondary keywords: Sintezna biologija;Disertacije;Synthetic biology;Methods;Signal transduction;Protein engineering;Logic;Protein interaction mapping;Proteolysis;Mammals;Cell line;Metode;Prenos signalov;Proteinski inženiring;Logika;Mapiranje proteinskih interakcij;Proteoliza;Sesalci;Celična linija;
Type (COBISS): Dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Medicinska fak.
Pages: XI, 75, VII f.
ID: 11224153