Utjecaj točkastih mutacija u genu antiCas i proteina HtpG na aktivnost sustava CRISPR-Cas u bakteriji Escherichia coli

master's thesis

• Title (english): The effects of point mutations in the antiCas gene and the HtpG protein on CRISPR-Cas system activity in Escherichia coli
• Author: Mirta Žagar
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Committee member: Nenad Malenica (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2024-02-23, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sustav CRISPR-Cas je prokariotski adaptivni obrambeni mehanizam protiv stranih genetičkih elemenata kao što su plazmidi i bakteriofagi. Ovaj sustav sastoji se od lokusa CRISPR koji sadrži segmente strane DNA (razmaknice) okružene palindromskim ponavljanjima te pridruženih gena cas koji sudjeluju u svim fazama obrane. Regulacija sustava CRISPR-Cas je kompleksna i nije još u potpunosti razjašnjena, a najviše je istražena u bakteriji Escherichia coli soja K-12. Neki od poznatih regulatora sustava CRISPR-Cas u bakteriji E. coli su šaperon HtpG, transkript antiCas i temperatura. U ovom radu istražen je utjecaj transkripta antiCas i njegovih točkastih mutanata u obrani bakterija od infekcije fagom λvir kada su nadeksprimirani s plazmida na 31°C. Osim toga, istražen je utjecaj nedostatka transkripta antiCas na aktivnost sustava CRISPR-Cas pri infekciji bakterija fagima na temperaturama od 31°C, 34,5°C te 37°C. Dobiveni rezultati ukazuju na negativan utjecaj transkripta antiCas i njegovih mutanata na aktivnost sustava CRISPR-Cas samo kad su eksprimirani u suvišku. Nadalje, pokazano je da transkript antiCas nema utjecaj na temperaturno ovisnu imunost bakterija na infekciju fagima, već da ona ovisi jedino o pomaku aminokiseline Trp-406 u proteinu Cas3.

Abstract (english)

The CRISPR-Cas system is a prokaryotic adaptive defense mechanism against foreign genetic elements such as plasmids and bacteriophages. This system consists of a CRISPR locus containing fragments of foreign DNA (spacers) surrounded by palindromic repeats, and the associated cas genes involved in all phases of defense. The regulation of the CRISPR-Cas system is very complex and not yet fully understood, and it has mainly been studied in the Escherichia coli strain K-12. Some of the known regulators of the CRISPR-Cas system in E. coli are the chaperone HtpG, the antiCas transcript, and temperature. In this study, the influence of the overexpression of the antiCas transcript and its point mutants from the plasmid on the defense of bacteria against λvir infection at 31°C was investigated. Also, the effect of the lack of the antiCas transcript on the activity of the CRISPR-Cas system against phage infection was investigated at temperatures of 31°C, 34.5°C, and 37°C. The obtained results indicate that the antiCas transcript and its mutants have a negative role on the activity of the CRISPR-Cas system only when they are overexpressed. Furthermore, it was shown that the antiCas transcript has no effect on the temperature-dependent immunity of the bacteria against phage infection, but that this depends only on the shift of the amino acid Trp-406 in the Cas3 protein.

• Keywords: temperatura; CRISPR-Cas; transkript antiCas; konformacija
• Keywords (english): temperature; CRISPR-Cas; antiCas transcript; conformation
• Language: croatian
• URN:NBN: urn:nbn:hr:217:763009
• Study programme: Title: Molecular Biology-University graduate programme; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra molekularne biologije (sveučilišni/a magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-02-23
• Created on: 2024-03-06 11:11:55