Određivanje uspješnosti genskog ciljanja u kvascu Kluyveromyces marxianus

master's thesis

• Title (english): Determination of gene targeting fidelity in yeast Kluyveromyces marxianus
• Author: Karla Johman
• Mentor: Ivan-Krešimir Svetec (mentor)
• Committee member: Igor Stuparević (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: 2020-09-29, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Kvasac Kluyveromyces marxianus termotolerantni je kvasac zanimljivih fizioloških svojstva zbog čega je interesantan i kao radni mikroorganizam za brojne biotehnološke procese. U ovome radu, određena je uspješnost genskog ciljanja u konstruiranom soju kvasca K. marxianus. U prvom koraku ovaj kvasac transformiran je linearnim fragmentom NatBAV koji sadrži selektivni biljeg coNat odgovoran za rezistenciju na antibiotik nurserotricin, a čija je integracija u genom potvrđena hibridizacijom po Southern-u. U drugom koraku soj koji je u genomu sadržavao fragment NatBAV, transformiran je linearnim fragmentom HygBAV koji sadrži gen coHyg odgovoran za rezistenciju na antibiotik higromicin, a s obje strane selektivnog biljega nalazile su se regije homologne fragmentu NatBAV. Kao rezultat uspješnog genskog ciljanja dolazi do zamjene genomske regije sekvencom na transformirajućoj DNA procesom homologne rekombinacije. Nakon selekcije na podlozi s antibiotikom higromicinom, dobiveni transformanti replicirani su na podlogu s nurserotricinom te je u 70 % transformanata utvrđena zamjena gena coNat genom coHyg. Ova uspješnost je postignuta s relativno dugim bočnim homologijama (1,4 i 2,5 kb), a na temelju rezultata je ustanovljeno da je gensko ciljanje u kvascu K. marxianus znatno uspješnije nego u većini drugih vrsta nekonvencionalnih kvasaca.

Abstract (english)

The yeast Kluyveromyces marxianus is a thermotolerant yeast with interesting physiological properties that make it interesting producer microorganism for different biotechnical processes. In this work, gene targeting fidelity in the constructed strain of yeast K. marxianus was determined. In the first step, this yeast has been transformed with the linear NatBAV DNA fragment containing a selectable coNat marker responsible for resistance to the antibiotic nourseothricin, whose random integration in the genome was confirmed by Southern blotting. In the second step, strain that contained integrated NatBAV fragment in the genome has been transformed with the linear HygBAV fragment containing coHyg gene responsible for antibiotic resistance to hygromycin, with flanking regions homologous to the NatBAV fragment on either side of the selective marker. As a result of successful gene targeting, the genomic region is replaced with the sequence on the transforming DNA by homologous recombination. After selection on a medium with the antibiotic hygromycin, the obtained transformants have been replicated to the medium with nourseothricin and in 70 % of the transformants the coNat gene was replaced with the coHyg gene. This fidelity was achieved with relatively long lateral homologies (1,4 and 2,5 kb) and based on the results it was found that gene targeting in K. marxianus was significantly more successful than in most other nonconventional yeast species.

• Keywords: Kluyveromyces marxianus; gensko ciljanje; transformacija; homologna rekombinacija
• Keywords (english): Kluyveromyces marxianus; gene targeting; transformation; homologous recombination
• Language: croatian
• URN:NBN: urn:nbn:hr:159:010295
• 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: 2021-01-21 10:52:16