magistrsko delo
Tim Godec (Author), Jernej Ogorevc (Reviewer), Hrvoje Petković (Mentor)

Abstract

Razvoj celičnih tovarn na osnovi bakterij iz rodu Streptomyces je ključen za proizvodnjo novih in že poznanih biološko aktivnih učinkovin. S postopno delecijo večjih delov genoma lahko razvijemo učinkovito platformo za heterologno izražanje sekundarnih metabolitov. Klasične metode za manipulacije genoma v streptomicetah so zahtevne in časovno potratne. V tem delu, smo z uporabo CRISPR-Cas9 sistema izvedli delecijo genoma bakterije Streptomyces rimosus v velikosti 145 kb. Z bioinformacijsko analizo smo izbrali tarčno regijo genoma, ustrezne homologne regije in vodilne RNA. Z metodama PCR s prekrivanjem in SLiCE kloniranje smo sestavili plazmidni konstrukt za delecijo s CRISPR-Cas9. Konstrukt smo s konjugacijo vnesli v S. rimosus in testirali delecijo v pozitivnih klonih z metodo PCR in sekvenciranjem. Identificirali smo številne seve s pravilno delecijo izbranega fragmenta DNK v velikosti 145 kb.

Keywords

CRISPR;delekcija;redukcija genoma;Streptomyces rimosus;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [T. Godec]
UDC: 602.3:579.873.7:602.6:579.8(043.2)
COBISS: 76574723 Link will open in a new window
Views: 634
Downloads: 2
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Other data

Secondary language: English
Secondary title: Large genomic deletion in bacteria Streptomyces rimosus using CRISPR-Cas9
Secondary abstract: Developing a Streptomyces-based cell factory is key for the production of new or known biologically active compounds. With gradual deletion of large genomic regions one can develop an efficient platform for the heterologous expression of secondary metabolites. Gene manipulations of Streptomyces using classical approaches are demanding and time-consuming. In this work we used CRISPR-Cas9 to achieve a 145 kb genomic deletion in Streptomyces rimosus. With the use of bioinformatics we chose a target region with accompaning homologies and guide RNAs. We then used overlap PCR and SLiCE cloning to construct a plazmid for CRISPR-Cas9 driven deletion. The plasmid was introduced into S. rimosus with conjugation, then positive clones were tested for the deletion using PCR and sequencing. We have identified several isolates with correctly deleted DNA fragment of 145 kb in size.
Secondary keywords: genome reduction;genomic deletion;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 2024-09-09
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: IX, 56 f.
ID: 13403730