Development of methods for genetic manipulation of bacteria from order Bacillales

master's thesis

• Title (croatian): Razvoj metoda za genetičke manipulacije bakterija iz reda Bacillales
• Author: Tomislav Vološen
• Mentor: Anita Slavica (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2019-09-24, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Strains from order Bacillales have numerous industrial applications but due to the complexity and non-efficient transformation protocols have not reached their full implementation potential. This Master Thesis was focused on the transformation of selected strains from order Bacillales: Bacillus thuringiensis 407 cry-, Paenibacillus sp., Bacillus simplex, Lysinibacillus fusiformis M5, and Bacillus velezensis FZB42. In order to perform and follow their transformation, two vectors labelled through introduction of a green fluorescent protein gene were constructed by using Gibson assembly. Within transformation protocols special attention was paid to electroporation, suitable for the transformation of B. thuringiensis 407 cry-, and conjugation, optimal for the rest of the tested strains. Further, stability of the two constructed plasmids and two other, previously prepared plasmids, used in the transformation of B. thuringiensis 407 cry-, was tested by using fluorescence and resistance to antibiotics (kanamycin and chloramphenicol). In addition, plasmid gene expression was followed during growth of B. thurin-giensis 407 cry- by stereomicroscopy, root colonization assay, and confocal laser scanning microscopy. Based on obtained results it can be concluded that successfully transformed B. thuringiensis 407 cry- is good candidate for plant-growth-promoting bacterium.

Abstract (english)

Vrste iz roda Bacillales imaju široku primjenu u industrijskoj proizvodnji, ali zbog kom-pleksnosti i neučinkovitosti protokola za njihovu transformaciju još uvijek nisu dosegli svoj puni in-dustrijski potencijal. Ovaj je diplomski rad bio usmjeren na transformaciju odabranih vrsta iz reda Ba-cillales: Bacillus thuringiensis 407 cry-, Paenibacillus sp., Bacillus simplex, Lysinibacillus fusiformis M5 i Bacillus velezensis FZB42. Primjenom Gibsonove metode konstruirana su dva vektora, u koje je kao marker uveden gen za zeleni fluorescirajući protein. Ova su dva vektora korištena za optimiranje protokola za transformaciju pobrojenih vrsta, u okviru kojeg je posebna pozornost posvećena postupku elektroporacije, koji se pokazao učinkovitim za transformaciju B. thuringiensis 407 cry-, i konjugacije, koji je bio optimalan za preostale odabrane vrste iz ovog reda. Nadalje, stabilnost dvaju konstruiranih plazmida kao i dva prethodno pripremljena plazmida, koji su se koristili za transformaciju B. thurin-giensis 407 cry-, testirana je uz primjenu metode za određivanje fluorescencije i rezistencije na antibi-otike (kanamicin i kloramfenikol). Dodatno, praćena je ekspresija gena plazmida tijekom rasta B. thu-ringiensis 407 cry- metodama stereomikroskopije, kolonizacije korijena biljke i konfokalnom la-serskom skenirajućom mikroskopijom. Na temelju dobivenih rezultata može se zaključiti da je uspješno transformirani B. thuringiensis 407 cry- dobar kandidat za grupu bakterija koje pospješuju rast biljaka.

• Keywords: Bacillales; Bacillus thuringiensis; Gibson assembly; transformation; plant-growth-promoting bacterium
• Keywords (english): Bacillales; Bacillus thuringiensis; Gibsonova metoda; transformacija; bakterije koje pospješuju rast biljaka
• Language: english
• URN:NBN: urn:nbn:hr:159:455900
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-12-13 13:25:51