Određivanje količine proteina Cas3 u mutantima Δhns i ΔhtpG bakterije Escherichia coli u različitim uvjetima rasta

master's thesis

• Title (english): Determining the quantity of the Cas3 protein in Δhns and ΔhtpG mutants in Escherichia coli under different growth conditions
• Author: Marin Macuka
• Mentor: Ivana Ivančić Baće (mentor)
• Mentor: Petra Peharec Štefanić (mentor)
• Mentor: Kristina Majsec (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Željka Vidaković Cifrek (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sustav CRISPR-Cas je obrambeni mehanizam mnogih prokariota od infekcija stranim nukleinskim kiselinama poput virusa i plazmida. Lokus CRISPR sastoji se od niza palindromskih ponavljajućih sekvenci razdvojenih razmaknicama, podrijetlom iz faga ili plazmida. Uz lokus CRISPR nalaze se pridruženi geni cas, čijom ekspresijom nastaju proteini Cas koji sudjeluju u obrambenom mehanizmu. Lokus se prepisuje u dugu prekursorsku RNA koja se procesira u manje RNA zvane CRISPR-RNA (crRNA) koje se spajaju s jednim ili više proteina Cas. Taj kompleks pronalazi strane DNA temeljem komplementarnosti sekvenci, te nakon uspješnog prepoznavanja, nastaje R-omča. Tu strukturu prepoznaje protein Cas3 koji nakon vezanja cijepa stranu DNA. Nekoliko čimbenika utječe na aktivnost sustava CRISPR-Cas u bakteriji E. coli: temperatura, protein H-NS, te šaperon HtpG. Pri 37 0C uočen je gubitak aktivnosti proteina Cas3 iz nepoznatih razloga. U ovom radu istraživana je količina proteina Cas3 u sojevima uzgojenim pri temperaturi od 30 0C i 37 0C, te u sojevima s mutiranim genima hns, htpG ili oba. Analizom količine proteina Cas3 u različitim mutantama bakterije E. coli, nije uočena razlika u količini proteina Cas3 u ovisnosti o temperaturi rasta, već u ovisnosti o genotipu mutanta i to mutaciji hns.

Abstract (english)

The CRISPR-Cas system is a defense mechanism employed by many prokaryotes against foreign nucleic acids infections, e.g. viruses and plasmids. The locus CRISPR consists of a series of short palindromic repeats separated by spacers derived from foreign DNA. Near the CRISPR array are cas genes which encode Cas proteins that participate in the defense mechanism. The array is transcribed into a long precursor RNA that is processed into smaller RNA called CRISPR-RNA (crRNA) that fuse with one or more Cas proteins. This complex finds foreign DNA through complementary base pairing, which if successful, generates an R-loop. This structure is recognized by Cas3 which cleaves the foreign DNA after binding. Several factors affect the activity of CRISPR-Cas in E. coli - temperature, protein H-NS, and chaperon HtpG. A loss of Cas3 activity was observed at 37 0C due to unknown reasons. In this work, the amounts of Cas3 were determined in strains grown at 30 0C and 37 0C, and also in strains that contained mutation in hns, htpG gene or both. Differences in Cas3 amounts in different E. coli mutants weren't dependent on the growth temperature, but rather on the mutant's genotype, specifically hns mutation.

• Keywords: CRISPR-Cas; Cas3; temperatura; E. coli
• Keywords (english): CRISPR-Cas; Cas3; temperature; E. coli
• Language: croatian
• URN:NBN: urn:nbn:hr:217:845868
• Study programme: Title: Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra edukacije biologije (magistar/magistra edukacije biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2020-09-27
• Created on: 2019-11-29 10:36:54