Analiza transformanata kvasca Scheffersomyces stipitis metodom hibridizacije DNA po Southernu

undergraduate thesis

• Title (english): Analysis of the Scheffersomyces stipitis transformants by Southern blot hybridization
• Author: Martina Merdžo
• Mentor: Ivan-Krešimir Svetec (mentor)
• Mentor: Marina Svetec Miklenić (pomoć pri izradi, neposredni voditelj)
• 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 analiza nekonvencionalnog kvasca Scheffersomyces stipitis, prethodno transformiranog s dvije različite linearne molekule DNA, kako bi se dokazala ilegitimna integracija ovih molekula u genom kvasca. Svaki transformirajući fragment nositelj je po jednog od dva gena potrebna za pretvorbu piruvata u vrijedni 2,3-butandiol te gena za rezistenciju na antibiotik, koji je korišten kao genetički biljeg za selekciju transformanata. Molekularna analiza provedena je metodom hibridizacije DNA po Southernu. Probe obilježene digoksigeninom za detekciju integriranih fragmenata sintetizirane su metodom PCR (eng. Polymerase Chain Reaction, lančana reakcija polimerazom). Probe su konstruirane tako da budu komplementarne samo kodirajućoj DNA (ORF, eng. Open Reading Frame, otvoreni okvir čitanja) gena za odgovarajuću antibiotičku rezistenciju na transformirajućim fragmentima. Analiza je pokazala da je u svim transformantima uspješno provedena integracija obje transformirajuće molekule DNA na nasumičnim mjestima u genomu kvasca.

Abstract (english)

The aim of this work was to analyse the non-conventional yeast Scheffersomyces stipitis, previously transformed with two different linear DNA molecules, to prove the illegitimate integration of these molecules into the yeast's genome. Each transforming DNA fragment contains one of two genes required for the conversion of pyruvate to a valuable 2,3-butanediol and an antibiotic resistance gene as a genetic marker for the selection of transformants. The molecular analysis was conducted by Southern blot hybridization. Digoxigenin labelled probes for detection of integrated fragments were synthesized by polymerase chain reaction (PCR). The probes were designed to be complementary only to coding DNA (Open Reading Frame, ORF) of antibiotic resistance genes on the transforming fragments. The analysis showed that the integration of two transforming DNA molecules at a random place in this yeast's genome was successful in all of the analysed transformants.

• Keywords: Scheffersomyces stipitis; metoda hibridizacije DNA po Southernu; ilegitimna integracija; nekonvencionalni kvasac; probe obilježene digoksigeninom
• Keywords (english): Scheffersomyces stipitis; Southern blot hybridization; illegitimate integration; nonconventional yeast; digoxigenin labelled probes
• Language: croatian
• URN:NBN: urn:nbn:hr:159:483065
• 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 12:06:58