Utjecaj brzine rasta na efikasnost adaptacije sustavom CRISPR-Cas u bakterija Escherichia coli različitih genetičkih pozadina

master's thesis

• Title (english): The influence of growth rate on the efficiency of the CRISPR-Cas mediated adaptation in Escherichia coli of different genetic backgrounds
• Author: Patricija Knechtl
• Mentor: Ivana Ivančić Baće (mentor)
• Mentor: Damjan Mitić (komentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Mirela Sertić Perić (član povjerenstva)
• Committee member: Draginja Mrvoš-Sermek (član povjerenstva)
• Committee member: Željka Soldin (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-10-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sustav CRISPR-Cas sastoji se od lokusa CRISPR koji zajedno s pridruženim genima cas
omogućuje adaptivnu imunost mnogim bakterijama i većini arheja protiv stranih genetičkih
elemenata. Lokus CRISPR sastoji se od brojnih ponavljajućih sekvenci DNA koje su
razdvojene kratkim razmaknicama strane DNA. Naivna adaptacija prva je faza zaštite u kojoj
se kratki fragmenti iz stranih organizama ugrađuju u lokus CRISPR kako bi se zaštitila stanica
od budućih napada istom stranom DNA. U ovom radu istražen je utjecaj ekspresije gena cas1 i
cas2 na brzinu rasta stanica, određena je faza rasta u kojoj dolazi do ugradnje nove razmaknice
te je istražena efikasnost adaptacije u tri različita bakterijska soja E. coli koji imaju tri različite
genetičke pozadine (BW25113, AB1157 i MG1655). Također, istraženo je kako količine
proteina Cas1 i Cas2 utječu na efikasnost naivne adaptacije (ugradnju razmaknice). Na temelju
analize dobivenih rezultata može se zaključiti kako se usporeni rast bakterijskih stanica javlja
zbog ugradnje razmaknice u lokus CRISPR, u ranoj log fazi rasta, samo u genetičkoj pozadini
BW25113. U bakterijskim sojevima koji su rasli sporo došlo je do ekspresije proteina Cas1 i
Cas2, dok geni rpoS i crl nemaju ulogu u izgradnji adaptivne imunosti procesom naivne
adaptacije.

Abstract (english)

The CRISPR-Cas system consists of the CRISPR locus, which, together with the CRISPRassociated (cas) genes, provides adaptive immunity to many bacteria and most archaea against
foreign genetic elements. The CRISPR locus consists of numerous repetitive DNA sequences
separated by short spacers that are derived from foreign DNA. Naïve adaptation is the first
phase of protection in which short fragments from foreign organisms are incorporated into the
CRISPR locus to protect the cell from future attacks by the same foreign DNA. In this research,
we investigated the influence of cas1 and cas2 gene expression on cell growth rate, we
determined in which growth phase a new spacer is incorporated and we investigated the
efficiency of adaptation in three different bacterial strains of E. coli with three different genetic
backgrounds (BW25113, AB1157 and MG1655). Also, we investigated how the amounts of
Cas1 and Cas2 proteins affect the efficiency of adaptation (spacer acquisition). Based on the
analysis of the obtained results, it can be concluded that the slowed growth of bacterial cells
occurs due to the insertion of a spacer into the CRISPR locus in the early log phase of growth
only in the BW25113 genetic background. In bacterial strains that grew slowly, Cas1 and Cas2
proteins were expressed. Genes rpoS and crl have no role in building CRISPR immunity
through the process of naïve adaptation.

• Keywords: naivna adaptacija; ugradnja razmaknica; lokus CRISPR; rpoS; crl; E. coli
• Keywords (english): naive adaptation; spacer integration; locus CRISPR; rpoS; crl; E. coli
• Language: croatian
• URN:NBN: urn:nbn:hr:217:188517
• Study programme: Title: Integrated undergraduate and graduate university study of Biology and Chemistry Education; specializations in: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Course: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra edukacije biologije i kemije (magistar/magistra edukacije biologije i kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-08-25
• Created on: 2022-11-03 13:08:47