Konstrukcija plazmida koji omogućuje pojačanu ekspresiju konstitutivno aktivnog alela YAP1C620F

undergraduate thesis

• Title (english): Construction of the overexpression plasmid carrying constitutively active YAP1C620F allele
• Author: Lada Ćurković
• Mentor: Anamarija Štafa (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2019-09-09, 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: alel YAP1C620F; gen YAP1; kvasac Saccharomyces cerevisiae; mutageneza; točkasta mutacija
• Keywords (english): YAP1C620F allele; YAP1 gene; mutagenesis; point mutation; yeast Saccharomyces cerevisiae
• Language: croatian
• URN:NBN: urn:nbn:hr:159:714419
• Study programme: Title: 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 resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2019-09-10 11:38:25