Razvoj orodij CRISPR-Cas pri bakteriji Streptomyces rimosus

Reberšek, Roman (avtor), Petković, Hrvoje (mentor)

Bakterije iz rodu Streptomyces so industrijsko in medicinsko pomembne proizvajalke biološko aktivnih učinkovin. Čeprav so za njihov genski inženiring že razvite klasične metode kloniranja na osnovi homologne rekombinacije, prinašajo novejše metode, kot na primer z encimi Cas, nekatere potencialne izboljšave in zato smiselno dopolnjujejo nabor molekularnih orodij tudi za ta rod. Iz nabora nukleaz Cas smo izbrali dve, ki sta se nam po pregledu literature zdeli najbolj zanimivi. Gre za nukleazi Cas9 iz Streptococcus pyogenes in Cas12a iz Francisella novicida. Za njuno izražanje smo sestavili štiri segregacijsko nestabilne plazmide, pri čemer smo z vsako nukleazo poizkusili izvesti delecijo istih dveh tarč v genomu Streptomyces rimosus, ki je industrijsko pomemben producent oksitetraciklina. Prva tarča je bila regija genov oxyA in oxyB, ki nosita zapisa za ključna encima poliketidne sintaze na poti biosinteze oksitetraciklina. Druga tarča pa je bil gen za sekretorno tripeptidil aminopeptidazo, eno izmed bolj zastopanih zunajceličnih proteaz pri bakteriji S. rimosus. Plazmidne konstrukte s komponentami sistema CRISPR-Cas smo pripravili z uporabo modernih metod kloniranja na osnovi homologij, kot so PCR s prekrivanjem, NEBuilder® HiFi/Gibson Assembly® in SLiCE kloniranje. Plazmidne konstrukte smo z elektroporacijo najprej vnesli v bakterijo Escherichia coli, nato pa iz nje s konjugacijo v S. rimosus. Po različnih metodah gojenja smo potrdili uspešne delecije obeh tarč s PCR in s sekvenciranjem pomnoženega fragmenta DNA mutant S. rimosus.

• Jezik: Slovenski jezik
• Ključne besede: genski inženiring, genska modifikacija, CRISPR, Cas9, Cas12, aktinomicete, Streptomyces rimosus, oksitetraciklin, sekretorna tripeptidil aminopeptidaza
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Kraj izida: Ljubljana
• Založnik: [R. Reberšek]
• Leto izida: 2021
• PID: 20.500.12556/RUL-129482
• UDK: 602.6:579.873.7
• COBISS.SI-ID: 74967043
• Datum objave v RUL: 02.09.2021

REBERŠEK, Roman, 2021, Razvoj orodij CRISPR-Cas pri bakteriji Streptomyces rimosus [na spletu]. Magistrsko delo. Ljubljana : R. Reberšek. [Dostopano 3 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=129482

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of CRISPR-Cas tools in bacteria Streptomyces rimosus

Izvleček

Bacteria of the genus Streptomyces are industrially and medically important producers of biologically active compounds. Although classical cloning methods based on homologous recombination have already been developed for their genetic engineering, newer methods such as those with the Cas enzymes bring some potential improvements and therefore sensibly complement the set of molecular tools for this genus. We chose to use two different Cas nucleases that we found the most interesting after reviewing available literature: Cas9 from Streptococcus pyogenes and Cas12a from Francisella novicida. To express them, we assembled four segregationally unstable plasmids, using each nuclease to attempt a deletion of the same two genes on the genome of Streptomyces rimosus, an industrially important producer of oxytetracycline. The first target was the region of the oxyA and oxyB genes, which encode key enzymes of the polyketide synthase in the pathway of oxytetracycline biosynthesis. The second target was the gene for secretory tripeptidyl aminopeptidase, one of the more abundant extracellular proteases in S. rimosus. Plasmid constructs with CRISPR-Cas system components were prepared using modern cloning methods based on homologies such as overlap PCR, NEBuilder® HiFi/Gibson Assembly® and SliCE cloning. Plasmid constructs were first transformed into Escherichia coli by electroporation, followed by conjugation from E. coli into S. rimosus. After applying diverse culturing methods, successful deletions of both target genes were confirmed by PCR and sequencing of the amplified DNA fragment of S. rimosus mutants.

• Ključne besede: genetic engineering, gene modification, CRISPR, Cas9, Cas12, actinomycetes, Streptomyces rimosus, oxytetracycline, secretory tripeptydil aminopeptidase