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undergraduate thesis
Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. urn:nbn:hr:159:028349
Mihaljević, Ante
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biology and Microbial Genetics

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

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Mihaljević, A. (2019). Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR (Undergraduate thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:028349

Mihaljević, Ante. "Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR." Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. https://urn.nsk.hr/urn:nbn:hr:159:028349

Mihaljević, Ante. "Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR." Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. https://urn.nsk.hr/urn:nbn:hr:159:028349

Mihaljević, A. (2019). 'Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR', Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 15 July 2024, https://urn.nsk.hr/urn:nbn:hr:159:028349

Mihaljević A. Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR [Undergraduate thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2019 [cited 2024 July 15] Available at: https://urn.nsk.hr/urn:nbn:hr:159:028349

A. Mihaljević, "Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR", Undergraduate thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2019. Available at: https://urn.nsk.hr/urn:nbn:hr:159:028349

Metadata
TitleKloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR
Title (english)Cloning of DNA fragment from lambda genome into plasmids pRS40 coKanR, pRS40 coHyrR and pRS40 coNrsR
AuthorAnte Mihaljević
MentorIvan-Krešimir Svetec (mentor)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Biochemical Engineering)
(Laboratory for Biology and Microbial Genetics)
Zagreb
Defense date and country2019-06-04, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
Cilj rada bio je klonirati odabrani fragment genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR, i tako konstruirati plazmide pRS40 coKanR+L, pRS40 coHyrR+L i pRS40 coNrsR+L koji će se u budućnosti koristiti u eksperimentalnom sustavu za istraživanje učestalosti homologne rekombinacije u nekonvencionalnim (ne-Saccharomyces) kvascima. U svrhu konstrukcije navedenih plazmida, odabrani fragment genoma bakteriofaga lambda umnožen je lančanom reakcijom polimeraze (metoda PCR) te pocijepan restrikcijskom endonukleazom HindIII. Tako pripremljeni insert ligiran je s plazmidima koji su također linearizirani enzimom HindIII, a osim toga i defosforilirani. Za selekciju transformanata koji u plazmidima sadrže insert korištena je selekcija plavo-bijelo. Nakon transformacije dobiveno je nekoliko bijelih kolonija iz kojih je izolirana plazmidna DNA, međutim, na temelju rezultata gel-elektroforeze pokazano je da plazmidi izolirani iz transformanata ne sadrže insert. Pretraživanjem literature i analizom funkcije svakog gena koji se nalazi na umnoženom fragmentu genoma bakteriofaga lambda utvrđeno je da se na njemu nalazi gen koji kodira za peptid dugačak 86 aminokiselina koji prilikom indukcije profaga ubija stanice domaćina. Stoga je najvjerojatnije prisutnost ovog gena onemogućila uspješno kloniranje željenog fragmenta DNA.
Abstract (english)
The aim of this paper was to clone the selected fragment of lambda genome into plasmids pRS40 coKanR, pRS40 coHyrR and pRS40 coNrsR, and thus construct plasmids pRS40 coKanR+L, pRS40 coHyrR+L and pRS40 coNrsR+L which will be used in the experimental system for studying the efficiency of homologous recombination in unconventional (non-Saccharomyces) yeast. For the purpose of constructing said plasmids, the selected fragment of lambda genome was amplified by polymerase chain reaction (PCR method) and cleaved by restriction endonuclease HindIII. The thus-prepared insert is ligated with plasmids which are also linearized with the enzyme HindIII and, furthermore, dephosphorylated. Selection of transformants that contain plasmids with insert was carried with blue-white screening. After the transformation, several white colonies were obtained from which plasmid DNA was isolated, however, based on the results of gel electrophoresis, it was shown that plasmids isolated from transformants did not contain an insert. By searching the literature and analyzing the function of each gene located on a multiplied fragment of lambda genome, it has been found that there is a gene encoding the 86 amino acids long peptide responsible for lysis of the host cells after prophage induction. Therefore, most likely the presence of this gene did not allow successful cloning of the desired DNA fragment.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:159:028349
Study programmeTitle: Biotechnology
Study programme type: university
Study level: undergraduate
Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
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Created on2019-06-07 11:25:00