Kloniranje dijela genoma bakteriofaga lambda u plazmide pRS40 coKanR, pRS40 coHyrR i pRS40 coNrsR

undergraduate thesis

• Title (english): Cloning of DNA fragment from lambda genome into plasmids pRS40 coKanR, pRS40 coHyrR and pRS40 coNrsR
• Author: Ante Mihaljević
• Mentor: Ivan-Krešimir Svetec (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2019-06-04, 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: ne-Saccharomyces kvasci; homologna rekombinacija; kloniranje dijela genoma bakteriofaga lambda
• Keywords (english): cloning of DNA fragment from lambda genome; homologous recombination; non-Saccharomyces yeast
• Language: croatian
• URN:NBN: urn:nbn:hr:159:028349
• Study programme: Title: 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 resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-06-07 11:25:00