magistrsko delo
David Škrinjar (Author), Uroš Petrovič (Reviewer), Neža Čadež (Mentor), Luka Kranjc (Co-mentor)

Abstract

Razvoj molekularnih orodij in metod genskega inženiringa ima ključno vlogo pri razvoju novih gostiteljskih organizmov in njihovem prenosu v biotehnološke procese. Do sedaj sta bila industrijsko najpomembnejša mikrobna gostitelja bakterija Escherichia coli ter kvasovka Saccharomyces cerevisiae, vendar se vedno več pozornosti posveča tudi uporabi nekonvencionalnih vrst kvasovk. V sklopu naloge smo se posvetili vrsti Hanseniaspora uvarum, ki jo najpogosteje najdemo na grozdnih jagodah in ima pomembno vlogo pri fermentaciji mošta in posledično lahko vpliva na kakovost vina. V naši raziskavi smo v kvasovki S. cerevisiae sestavili dva plazmida, ki sta vsebovala 700-1000 bp dolgi regiji, homologni ribosomalni RNA oziroma transpozona Ty3. To je omogočilo integracijo genske kasete v genom H. uvarum, katera je vsebovala selekcijski označevalec za odpornost proti higromicinu, ter zapis za zeleni fluorescenčni protein. Vsebovala je tudi elemente, ki omogočajo pomnoževanje plazmida tako v kvasovki S. cerevisiae, kot v bakteriji E. coli. Pripravljeni plazmid smo vnesli v H. uvarum z kombinirano metodo transformacije z uporabo litijevega acetata in elektroporacije, kjer se je genska kaseta preko homologne rekombinacije integrirala v genom. Ker je bila učinkovitost transformacije H. uvarum zelo nizka, smo metodo transformacije poskusili izboljšati, vendar nam to ni uspelo. Kljub temu smo uspeli s homologno rekombinacijo vstaviti in izraziti gen za zeleni fluorescenčni protein v genomu H. uvarum. To bi v prihodnosti lahko pripomoglo k označevanju sevov ter spremljanju celične lokalizacije oziroma rasti H. uvarum v ko-kulturah.

Keywords

molekularna orodja;genetsko spreminjanje;Hanseniaspora uvarum;nekonvencionalne kvasovke;Saccharomyces cerevisiae;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [D. Škrinjar]
UDC: 579.25:577.2.088
COBISS: 239320323 Link will open in a new window
Views: 163
Downloads: 37
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Other data

Secondary language: English
Secondary title: Development of molecular tools and methods for genetic modification of yeast Hanseniaspora uvarum
Secondary abstract: The development of molecular tools and methods for genetic modification of organisms plays a very important role in the production of new metabolites and development of new host organims and their transfer into biotechnological processes. Bacteria Escherichia coli and yeast Saccharomyces cerevisiae have so far featured as the most successful industrial hosts, but more attention is nowadays being paid to the use of non-conventional yeasts. We focused on the non-conventional yeast species Hanseniaspora uvarum, which is most often found on grape berries, where it also plays a very important role in the alcoholic fermentation and can consequently affect the quality of wine. Through our research, we asembled two plasmids in yeast S. cerevisiae with 700-1000 bp long homologous region of ribosomal RNA or transposon Ty3, which allowed integration of the gene cassettte into the H. uvarum genome. The assembled plasmid also carried sequences for a selection marker, green fluorescent protein and elements that allow plasmid replication in both S. cerevisae and E. coli. Plasmid was then inserted into H. uvarum by combined transformation with electroporation and lithium acetate, where the gene cassette was integrated into the genome by homologous recombination. Because of low transformation efficiency we tried to improve the transformation method for H. uvarum, but unfortunately we did not succeed,. However, we achieved insertion of the gene for green fluorescent protein into H. uvarum by homologous recombination and we also confirmed its successful expression. In the future, this approach could be utilized for strain labeling, cellular localization, as well as growth and viability monitoring.
Secondary keywords: molecular tools;genetic modification;Hanseniaspora uvarum;nonconventional yeasts;Saccharomyces cerevisiae;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za mikrobiologijo
Pages: 1 spletni vir (1 datoteka PDF (XI, 54 str., [5] f. pril.))
ID: 26538872