Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom

master's thesis

• Title (english): The improvement of the transformation procedure by electroporation for the yeast Scheffersomyces stipitis
• Author: Lada Ćurković
• Mentor: Anamarija Štafa (mentor)
• Committee member: Ivan Krešimir Svetec (član povjerenstva)
• 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: 2021-12-03, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Nekonvencionalni kvasac Scheffersomyces stipitis, izoliran je iz trulećeg drva i larvi insekata koji nastanjuju drvo. S. stipitis ima jednu od najviših sposobnosti izravne fermentacije ksiloze u etanol što je posebno zanimljivo pri proizvodnji bioetanola iz lignoceluloznih sirovina. Ovaj kvasac je prototrof, rezistentan je na antibiotike koji se često koriste kao selektivni biljezi pri transformaciji, nisu razvijeni visokoefikasni postupci za transformaciju te mu efikasnost transformacije iznosi 1∙10³ transformanata μgˉ¹. Cilj ovog rada bio je modificirati protokol za elektroporaciju kako bi se postigla viša efikasnost transformacije te odrediti uspješnost genskog ciljanja mehanizmom rekombinacije krajevi-van. Modificirani protokol za elektroporaciju omogućio je postizanje efikasnosti transformacije više od 1∙10⁵ transformanata μgˉ¹. Uspješnost genskog ciljanja, u sustavu u kojem se istraživala zamjena gena SsURA3 genom koji nosi rezistenciju na higromicin, koji se nalazi na disrupcijskoj kazeti, procijenjena je na manje od 0,2 % jer prilikom transformacije nije nastao nijedan transformant željenog fenotipa. Dodatno, istražena je i mogućnost primjene CRISPR/Cas sustava kako bi se povećala uspješnosti genskog ciljanju u kvascu S.stipitis.

Abstract (english)

Scheffersomyces stipitis is a non-conventional yeast known for its capacity to ferment xylose and convert it to ethanol which is useful in the production of bioethanol from lignocellulosic materials. S. stipitis is isolated from decaying wood and larvae of wood-inhabiting insects. This yeast species is prototroph and resistant to antibiotics frequently used as selection markers for transformation. Furthermore, there are no highly efficient transformation protocols resulting in transformation efficiency around 1∙10³ transformants μgˉ¹. The aim of this research was to determine the electroporation parameters that increase the efficiency of transformation and to determine the percentage of gene targeting by ends-out recombination. Modified electroporation protocol resulted in transformation efficiencies higher than 1∙10⁵ transformants μgˉ¹. The expected result of gene targeting by ends-out recombination was the replacement of SsURA3 gene with a hygromycin resistance gene on the disruption cassette and it was estimated to be lower than 0.2 % since none of the transformants showed the desired phenotype. Additionally, the use of CRISPR/Cas system for the improvement of gene targeting efficiency in S.stipitis was analyzed.

• Keywords: kvasac Scheffersomyces stipitis; transformacija; elektroporacija; gensko ciljanje; sustav CRISPR/Cas
• Keywords (english): yeast Scheffersomyces stipits; transformation; electroporation; gene targeting; CRISPR/Cas system
• Language: croatian
• URN:NBN: urn:nbn:hr:159:375037
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-12-07 12:49:20