diplomsko delo
Marija Zaharieva (Author), Hrvoje Petković (Mentor), Hrvoje Petković (Thesis defence commission member), Marko Blažič (Thesis defence commission member), Polona Jamnik (Thesis defence commission member), Mojca Narat (Thesis defence commission member), Marko Blažič (Co-mentor)

Abstract

Bakterija Bacillus subtilis je dober modelni mikroorganizem za proučevanje različnih procesov v celici, kot so metabolizem, genska regulacija in bakterijska diferenciacija. Na voljo imamo dobro razvite metode za gensko manipulacijo, zato je B. subtilis zelo privlačen kot gostiteljski sistem za uporabo metabolnega inženirstva. Uspešno izražanje genov in njihova regulacija sta namreč ključni za razvoj industrijskih sevov. Genska tehnologija nam omogoča vstavljanje novih genov, prekinitev neželjenih genov ter kontroliranje izražanja genov. Leta 2013 je bila razvita nova metoda za urejanje genoma CRISPR/Cas9 (clustered regularly interspaced palindromic repeats s Cas9 proteinom), ki temelji na uporabi sistema za obrambo pred virusnimi okužbami pri bakterijah. Je učinkovita, enostavna in poceni metoda za urejanje genoma. Omejitev CRISPR/Cas9 je v tem, da včasih ne želimo inducirati preloma DNA in njene inaktivacije oziroma modifikacije. Ampak želimo samo inhibirati izražanje gena. Za ta namen se uporablja metoda CRISPRi/dCas9, s katero se lahko regulira transkripcijo. Metoda je zelo uporabna v proučevanju in izražanju esencialnih genov. V nalogi smo uspeli eksperimentalno pokazati delovanje metode CRISPRi/dCas9 na primeru proteina GFP (green fluorescent protein) v B. subtilis. Encim dCas9 ni rezal gena, ki kodira GFP (kar bi se pokazalo kot popolna odsotnost zelene fluorescence), ampak je le zmanjšal njegovo izražanje (manjša intenziteta zelene fluorescence).

Keywords

biotehnologija;CRISPRi;dCas9;Bacillus subtilis;GFP;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [M. Zaharieva]
UDC: 602.6:579.852.11(043.2)
COBISS: 9364345 Link will open in a new window
Views: 689
Downloads: 200
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Other data

Secondary language: English
Secondary title: Application of CRISPRi technology in Bacillus subtilis
Secondary abstract: Bacillus subtilis is a very atractive model microorganism to study bacterial metabolism, gene regulation and bacterial differentiation. Several methods for genetic manipulation of Bacillus subtilis genome have been developed. Therefore, B. subtilis is a promising host for application of metabolic engineering approaches. Successful gene expression and regulation are crucial for industrial applications. Development of gene tools enables transfer of genes to new hosts, deletion of undesirable genes and regulation of gene expression. In 2013, a new genome editing method, CRISPR/Cas9, was developed, which is based on bacterial antiviral immune response. It is an effective, simple and cheap genome-editing method. Limitation for CRISPR/Cas9 method is that it causes a double strand break in DNA, which results in gene inactivation or modification. For purposes of gene inhibition, a modification in Cas9 was made and a novel CRISPRi/dCas9 method was developed. This method enables regulation of gene expression on transcription level. CRISPRi/dCas9 method is useful for studying expression of essential genes. In this work, we used CRISPRi/dCas9 method to downregulate expression of GFP in B. subtilis. We observed decrease in green fluorescence emission, thus lower expression of GFP was confirmed in the culture treated with CRISPRi/dCas9. Therefore, we proved that CRISPRi/dCas9 is a useful method for genetic manipulation of B. subtilis.
Secondary keywords: biotechnology;
Type (COBISS): Bachelor thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: VIII, 20 str., [3] str. pril.
ID: 11218617