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undergraduate thesis
Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. urn:nbn:hr:159:714419
Ćurković, Lada
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biology and Microbial Genetics

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

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Ćurković, L. (2019). Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F (Undergraduate thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:714419

Ćurković, Lada. "Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F." Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. https://urn.nsk.hr/urn:nbn:hr:159:714419

Ćurković, Lada. "Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F." Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. https://urn.nsk.hr/urn:nbn:hr:159:714419

Ćurković, L. (2019). 'Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F', Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 16 November 2024, https://urn.nsk.hr/urn:nbn:hr:159:714419

Ćurković L. Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F [Undergraduate thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2019 [cited 2024 November 16] Available at: https://urn.nsk.hr/urn:nbn:hr:159:714419

L. Ćurković, "Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F", Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2019. Available at: https://urn.nsk.hr/urn:nbn:hr:159:714419

Metadata
TitleKonstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F
Title (english)Construction of the overexpression plasmid carrying constitutively active YAP1C620F allele
AuthorLada Ćurković
MentorAnamarija Štafa (mentor)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Biochemical Engineering)
(Laboratory for Biology and Microbial Genetics)
Zagreb
Defense date and country2019-09-09, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
Zbog složene kemijske strukture, kvasac ne može fermentirati lignocelulozne sirovine te ih je potrebno hidrolizirati, no pri hidrolizi se uz fermentabilne šećere oslobađaju i inhibitori rasta (slabe kiseline i derivati furana). Cilj ovog rada bio je uvesti točkastu mutaciju C620F u gen YAP1 čijom translacijom nastaje konstitutivno aktivan oblik transkripcijskog faktora Yap1 koji uzrokuje povećanu otpornost stanica kvasca na visoke koncentracije furfurala i hidroksimetilfurfurala. Mutacija je uvedena u gen YAP1 na plazmidu pSP-YAP1 u dva koraka. Tijekom lančane reakcije polimerazom sintetizirana je linearna plazmidna DNA koja sadrži točkastu mutaciju u genu YAP1. Linearni produkt je potom tretiran smjesom enzima KLD koja se sastoji od kinaze, ligaze i DpnI te je transformirana bakterija E. coli elektroporacijom. Iz transformiranih bakterijskih stanica izolirana je plazmidna DNA te je restrikcijskom analizom i sekvenciranjem potvrđeno uspješno uvođenje točkaste mutacije i konstrukcija plazmida pSP-YAP1C620F.
Abstract (english)
Due to their complex chemical structure yeasts cannot directly ferment lignocellulosic materials. Therefore, the lignocellulosis materials need to be hydrolysed releasing fermentable sugars and growth inhibitors (weak acids and furan derivatives). The aim of this research was to introduce a point mutation C620F in the YAP1 gene creating constitutively active transcription factor Yap1C620F that increases resistance of yeasts to high concetrations of furfural and hydroxymethylfurfural. This point mutation was introduced in the YAP1 gene on pSP-YAP1 plasmid in two steps. A linear plasmid DNA, with YAP1C620F allele, was synthesized in polymerase chain reaction. The PCR product was then treated with KLD mix, an enzyme mixture containing kinase, ligase and DpnI and then E. coli was transformed by electroporation. Plasmid DNA was isolated from transformants and the introduction of point mutation and construction of the pSP-YAP1C620F plasmid were confirmed by restriction analysis and Sanger sequencing.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:159:714419
Study programmeTitle: Biotechnology
Study programme type: university
Study level: undergraduate
Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
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Created on2019-09-10 11:38:25