prikaz prve stranice dokumenta Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom
  Download
PDF 1.3 MB
master's thesis
Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2021. urn:nbn:hr:159:375037
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

Cite this document

Ćurković, L. (2021). Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:375037

Ćurković, Lada. "Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2021. https://urn.nsk.hr/urn:nbn:hr:159:375037

Ćurković, Lada. "Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2021. https://urn.nsk.hr/urn:nbn:hr:159:375037

Ćurković, L. (2021). 'Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 08 January 2025, https://urn.nsk.hr/urn:nbn:hr:159:375037

Ćurković L. Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2021 [cited 2025 January 08] Available at: https://urn.nsk.hr/urn:nbn:hr:159:375037

L. Ćurković, "Poboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2021. Available at: https://urn.nsk.hr/urn:nbn:hr:159:375037

Metadata
TitlePoboljšanje postupka transformacije kvasca Scheffersomyces stipitis elektroporacijom
Title (english)The improvement of the transformation procedure by electroporation for the yeast Scheffersomyces stipitis
AuthorLada Ćurković
MentorAnamarija Štafa (mentor)
Committee memberIvan Krešimir Svetec (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Biochemical Engineering)
(Laboratory for Biology and Microbial Genetics)
Zagreb
Defense date and country2021-12-03, 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
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:159:375037
Study programmeTitle: 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 resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0) icon
Created on2021-12-07 12:49:20