magistrsko delo
Staš Vrh (Author), Hrvoje Petković (Reviewer), Aleš Podgornik (Mentor)

Abstract

Zaradi svoje ozke vrstne specifičnosti bakteriofagi predstavljajo možno alternativo uveljavljenemu zdravljenju bakterijskih okužb pri ljudeh z antibiotiki. Bakteriofagi se že uporabljajo v agroživilstvu za nadzor patogenov. Poleg zdravljenja bakterijskih okužb in nadzora patogenov se razvijajo tudi nove aplikacije bakteriofagov, kot so na primer dostavni sistemi za dostavo zdravil in genov. Zaradi širokega nabora aplikacij v prihodnosti pričakujemo povečevanje potrebe po proizvodnji bakteriofagov. Zaključni procesi proizvodnje bakteriofagov običajno vključujejo metode s slabo učinkovitostjo in težavnim prenosom v večje merilo. Razvoj monolitnih kromatografskih nosilcev je omogočil uvedbo kromatografije v analitiko in zaključne procese proizvodnje bakteriofagov. PolyHIPE predstavljajo nov razred monolitnih kromatografskih nosilcev, ki pa še niso bili preizkušeni za ločbo bakteriofagov. Namen magistrskega dela je bil preveriti uporabnost različno funkcionaliziranih polyHIPE kromatografskih kolon za ločbo fagov in njihove DNA ter optimizirati kromatografsko ločbo. Z uporabo polyHIPE kromatografske kolone, funkcionalizirane s QA, smo iz bakterijskega lizata uspeli ločiti bakteriofaga T4 in T7 ter njuno genomsko DNA. Povratni tlak se tudi po nekaj zaporednih ciklih vezave in elucije ni bistveno povečal. Z optimizirano kromatografsko metodo smo analizirali sestavo bakterijskega lizata in spremljali njeno spreminjanje med procesom gojenja fagov. Kromatografska metoda se je izkazala za primerno za medprocesno spremljanje gojenja fagov. Ugotovili smo, da predobdelava bakterijskega lizata z obarjanjem s PEG ali povečanje koncentracije Na+ v nalagalni raztopini lahko izboljšata ločljivost kromatografske metode.

Keywords

bakteriofagi;fagna DNA;ionsko izmenjevalna kromatografija;polyHIPE;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [S. Vrh]
UDC: 602.3:578.347:543.544.14(043.2)
COBISS: 74505731 Link will open in a new window
Views: 272
Downloads: 63
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Other data

Secondary language: English
Secondary title: Chromatography of bacteriophages and bacteriophage DNA using polyHIPE monolithic chromatographic column
Secondary abstract: Due to their narrow host range, bacteriophages offer a possible alternative to conventional antibiotic treatment of bacterial infections in humans. Phage-based control of pathogens is now already being used in agro-food sector. Apart from infection treatment and pathogen control, new bacteriophage applications such as drug and gene delivery system are being developed. Due to wide range of applications, the demand for bacteriophage production is expected to increase in the future. Downstream processes in phage production usually employ methods with low efficiency and poor scalability. The development of monolithic chromatografic media enabled the introduction of chromatography in the phage downstream processing and analytics. polyHIPE are a new class of monolithic chromatographic media that have not yet been tested for phage separation. The purpose of the master's thesis was to test the usability of differently functionalised polyHIPE chromatographic columns for phage and phage DNA separation and to optimise the chromatographic method. Using QA functionalised polyHIPE chromatographic column, we were able to separate T4 and T7 phage and their genomic DNA from phage lisate. The backpressure did not increase significantly even after a few consecutive cycles of binding and elution. Using optimised chromatographic method we analyzed the composition of phage lysate and monitor it's changing during the phage cultivation. The chromatographic method proved to be suitable for in-process control of phage cultivation. We found that pretreatment of phage lysate with PEG precipitation or the increase of Na+ concentration in loading sample can improve the resolution of the chromatographic method.
Secondary keywords: bacteriophages;phage DNA;ion-exchange chromatography;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: XII, 70 f.
ID: 13285830