Konstrukcija i molekularna analiza soja kvasca Saccharomyces cerevisiae FF OPO8 yku70delta

undergraduate thesis

• Title (english): Construction and molecular analysis of yeast Saccharomyces cerevisiae strain FF OPO8 yku70delta
• Author: Antea Melvan
• Mentor: Marina Svetec Miklenić (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: 2023-07-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Cilj ovog rada bio je delecija gena YKU70 u kvascu Saccharomyces cerevisiae soja FF OPO8. Delecija je postignuta korištenjem delecijske kazete YKU70KanMX4 koja ima krajeve homologne DNA regijama koje se nalaze uzvodno i nizvodno od gena YKU70 u genomu kvasca, što omogućava njenu integraciju u ciljno mjesto putem mehanizma ends-out homologne rekombinacije. Također, delecijska kazeta sadrži i selektivni marker KanMX4 koji je omogućio selekciju transformanata uzgojem na podlozi koja sadrži antibiotik geneticin. DNA je zatim izolirana iz odabranih transformanata, a uspješnost izolacije potvrđena je gel elektroforezom. PCR analiza izolirane DNA provedena je upotrebom KanIN-F i KanIN-R početnica, te YKU70IN-F i YKU70IN-R početnica kako bi se potvrdila integracija delecijske kazete YKU70KanMX4 i delecija gena YKU70. Umnoženi fragmenti dobiveni PCR metodom analizirani su gel elektroforezom, čime je utvrđeno da su tri od četiri odabrana transformanta željenog genotipa.

Abstract (english)

The objective of this work was to delete the YKU70 gene in the yeast Saccharomyces cerevisiae strain FF OPO8. Deletion was achieved using YKU70KanMX4 deletion cassette with ends homologous to the DNA regions flanking the YKU70 gene which enables its integration at the target site through ends-out homologous recombination mechanism. Additionally, the cassette contains the KanMX4 selective marker which enabled the selection of transformants through cultivation on a medium containing the antibiotic geneticin. DNA was then isolated from selected transformants and the success of the isolation was confirmed by gel electrophoresis. PCR analysis of the isolated DNA was performed using KanIN-F and KanIN-R primers or YKU70IN-F and YKU70IN-R primers to verify the integration of the YKU70KanMX4 deletion cassette and the deletion of the YKU70 gene. The amplified fragments obtained from the PCR were analysed by gel electrophoresis, which determined that three out of four selected transformants had the desired genotype.

• Keywords: delecija gena; Saccharomyces cerevisiae; ends-out homologna rekombinacija; PCR
• Keywords (english): gene deletion; Saccharomyces cerevisiae; ends-out homologous recombination; PCR
• Language: croatian
• URN:NBN: urn:nbn:hr:159:484955
• 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: Open access
• Created on: 2024-06-17 12:13:30