magistrsko delo
Tajda Buh (Author), Martina Turk (Mentor), Vera Župunski (Thesis defence commission member), Marko Dolinar (Thesis defence commission member)

Abstract

Aureobasidium pullulans je filamentozna gliva s kvasno obliko. Zaradi sposobnosti preživetja v ekstremnih pogojih je zanimiva za proučevanje in uporabo v industriji. Cilj magistrskega dela je bil pripraviti triptofanski avksotrofni sev Aureobasidium pullulans s sistemom CRISPR-Cas9, razvitim za uporabo v Aspergillus nidulans in nekaterih drugih filamentoznih glivah. Prekiniti smo želeli gen ApTRP1 z zapisom za encim fosforibozilantranil izomerazo, ki je tretji encim v sintezni poti triptofana. S prekinitvijo sinteze te esencialne aminokisline smo onemogočili rast in preživetje celice v odsotnosti triptofana. S sistemom CRISPR-Cas9 smo inducirali dvojni prelom DNA v predelu promotorja pred genom ApTRP1. V postopku popravljanja, usmerjenega s homologno rekombinacijo, je celica v genom vnesla zaporedje z zapisom za odpornost proti bleomicinu. Izbitje gena ApTRP1 oz. vgradnjo bleomicinske kasete smo zasledovali s protiselekcijo s 5-fluoroantranilno kislino, reakcijo PCR in rastjo na ploščah s oz. brez triptofana. Prekinjen gen ApTRP1 smo potrdili le z rastjo mutant na selekcijskem gojišču s 5-fluoroantranilno kislino. S primerjavo rasti na gojišču s triptofanom in brez njega ter reakcijo PCR izbitja ApTRP1 nismo uspeli potrditi.

Keywords

kvasovke;Aureobasidium pullulans;urejanje genoma;sistem CRISPR-Cas9;triptofanski avksotrof;gen ApTRP1;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [T. Buh]
UDC: 577.21(043.2)
COBISS: 1538321091 Link will open in a new window
Views: 730
Downloads: 198
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Other data

Secondary language: English
Secondary title: Preparation of auxotrophic mutants of yeast-like fungus Aureobasidium pullulans with CRISPR-Cas9 technology
Secondary abstract: Aureobasidium pullulans is a yeast-like fungus. It thrives under extreme conditions and produces biotechnologically interesting products. The aim of the master's thesis was to prepare a tryptophan auxotrophic strain of Aureobasidium pullulans with the CRISPR-Cas9 system developed for genetic engineering of filamentous fungi. Our assignment was to knock-out the ApTRP1 gene. Its product is the third enzyme in the synthetic pathway of tryptophan. By interrupting the synthesis of this essential amino acid, the growth of the cell is inhibited in the absence of tryptophan. The CRISPR-Cas9 system induced a double-strand DNA break in the promoter region of the ApTRP1 gene. A double-strand break was repaired by homology-directed repair where a bleomycin cassette was integrated into A. pullulans genome. We used three different approaches to determine the knock-out of ApTRP1 gene: counterselection with 5-fluoroanranilic acid, PCR and the growth on plates with or without tryptophan. The gene knock-out was confirmed only with the growth on the plates with 5-fluoroanranilic acid. We did not confirm the mutants with the other two approaches.
Secondary keywords: Aureobasidium pullulans;CRISPR-Cas9;tryptophan auxotroph;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 77 str.
ID: 11209685