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master's thesis
Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli
Zagreb: University of Zagreb, Faculty of Science, 2017. urn:nbn:hr:217:798087
Petanjek, Valentina
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

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Petanjek, V. (2017). Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:798087

Petanjek, Valentina. "Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli." Master's thesis, University of Zagreb, Faculty of Science, 2017. https://urn.nsk.hr/urn:nbn:hr:217:798087

Petanjek, Valentina. "Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli." Master's thesis, University of Zagreb, Faculty of Science, 2017. https://urn.nsk.hr/urn:nbn:hr:217:798087

Petanjek, V. (2017). 'Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli', Master's thesis, University of Zagreb, Faculty of Science, accessed 21 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:798087

Petanjek V. Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2017 [cited 2024 December 21] Available at: https://urn.nsk.hr/urn:nbn:hr:217:798087

V. Petanjek, "Uloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2017. Available at: https://urn.nsk.hr/urn:nbn:hr:217:798087

Metadata
TitleUloga nukleazne aktivnosti enzima RecBCD u naivnoj i pripremljenoj ugradnji razmaknica i vijabilnosti bakterije Escherichia coli
Title (english)The role of nuclease activity of the RecBCD enzyme in naive and primed adaptation and viability in Escherichia coli
AuthorValentina Petanjek
MentorIvana Ivančić Baće (mentor)
Committee memberMorana Dulić (predsjednik povjerenstva)
Committee memberIvana Ivančić Baće (član povjerenstva)
Committee memberNenad Judaš (član povjerenstva)
Committee memberIva Juranović Cindrić (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2017-02-23, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
Sustav CRISPR-Cas (od eng. clustered regularly interspaced short palindromic repeats i CRISPR associated) bakterijski je mehanizam obrane od stranih genetičkih elemenata. Sustav se sastoji od lokusa CRISPR u koji se ugrađuju razmaknice stranog podrijetla između ponavljajućih sljedova, te pridruženih proteina Cas. Sustav CRISPR-Cas predstavlja adaptivni imunološki odgovor koji se odvija u tri koraka. U prvom koraku zvanom adaptacija dolazi do ugradnje strane DNA u obliku razmaknice i sinteze dodatnog ponavljajućeg slijeda. Adaptacija se odvija, ovisno o proteinima Cas koji u njoj sudjeluju, na dva načina - kao naivna ili pripremljena. Proteini Cas1 i Cas2 su neophodni u oba načina. U ovom radu istražen je utjecaj nukleazne aktivnosti enzima RecBCD (mutanti recB i recD) na naivnu i pripremljenu ugradnju, te na vijabilnost, odnosno stabilnost plazmida u bakteriji Escherichia coli. Potvrdili smo ključnu ulogu nukleazne aktivnosti enzima RecBCD u naivnoj adaptaciji. U pripremljenoj adaptaciji nukleazna aktivnost enzima RecBCD nije neophodna, ali efikasnost ugradnje razmaknice je znatno manja i ovisi o broju stanica što nije slučaj u stanicama divljeg tipa. Ekspresija gena cas1 i cas2 s plazmida nije utjecala na vijabilnost istih mutanata, ali je za 1000 puta smanjila udio stanica s plazmidom u dvostrukom mutantu recD recJ. Genetičkom analizom pokazali smo da gubitak plazmida ovisi o kompleksu Cas1-Cas2. Diskutirani su mogući razlozi ovog efekta.
Abstract (english)
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated) system is a bacterial defence mechanism against foreign genetic elements. The system is comprised of CRISPR locus in which the foreign genetic sequences are incorporated, and of Cas associated proteins. CRISPR-Cas is an adaptive immunity that takes place in three steps. In the first step termed adaptation, the foreign DNA is inserted into the locus in form of a spacer and additional repeat is synthesised. Adaptation can be naïve or primed, depending on participating Cas proteins. Cas1 and Cas2 proteins are essential in both forms of adaptations. In this research we investigated the role of the nuclease activity of the RecBCD enzyme (recB and recD mutants) on naïve and primed adaptation, and also on viability, or plasmid stability in Escherichia coli. We confirmed a key role of the nuclease activity of the RecBCD enzyme in naïve adaptation. In primed adaptation, the nuclease activity of the RecBCD enzyme is not essential, but the efficiency of spacer integation is much lower and is dependent on the cell number which is not the case in wild type cells. Expression of the cas1 and cas2 genes from the plasmid did not affect the viability of the same mutants, but it caused a plasmid decrease of 1000 fold in double mutant recD recJ. Genetic analysis showed that the reason of this decrease is dependent on the Cas1-Cas2 complex. Possible reasons of this effect are discussed.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:798087
Study programmeTitle: Graduate university studies in chemistry; specializations in: Research programme
Course: Research programme
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2017-03-09 12:33:40