magistrsko delo
Brigita Valentinčič (Author), Anja Klančnik (Reviewer), Polonca Štefanič (Mentor)

Abstract

Značilnost divjih sevov Bacillus subtilis je pogosto slabša naravna kompetenca oziroma nekompetenca za prevzem DNA. Za namen preučevanja takšnih sevov je potrebno razviti orodja genetske manipulacije, s katerimi lahko dosežemo vgradnjo željenih odsekov DNA. Eno od možnosti priprave rekombinantnih sevov predstavlja metoda elektroporacije, ki je bila predmet raziskav našega magistrskega dela. Izvedli smo številne poskuse optimizacije metode, pri čemer smo testirali različne načine priprave elektrokompetentnih celic in parametre elektroporiranja. Začetni poskusi elektroporacije naravno kompetentnih sevov B. subtilis 168 trpC2 in PS-216 wt so bili uspešni, elektroporacija naravno nekompetentnih sevov pa se je izkazala za izredno težavno, saj nam metode ni uspelo optimizirati. Iz tega razloga smo v eksperimentalno delo uvedli pripravo rekombinantnih sevov s fagno transdukcijo. Uspelo nam je pripraviti mutante naravno nekompetentnih sevov, ki smo jih nato uporabili pri raziskavi izmenjave DNA med različno sorodnimi sevi pri rojenju na poltrdnem mediju B. Seve smo označili z genom za antibiotično odpornost in fluorescentnim markerjem. Testirali smo prenos DNA med tremi kombinacijami nesorodnih (»non-kin«) sevov in med izogeno kombinacijo. Do povišanega prenosa DNA je prišlo pri eni izmed testiranih »non-kin« kombinacij. Pri izogeni kombinaciji do prenosa DNA ni prišlo. Ugotovitev nakazuje na možnost horizontalnega genskega prenosa med sevi zaradi sorodstvenega razlikovanja in posledične indukcije kompetence pri sicer naravno nekompetentnih sevih.

Keywords

Bacillus subtilis;elektroporacija;nekompetentni sevi;sorodstveno razlikovanje;horizontalen genski prenos;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [B. Valentinčič]
UDC: 602.621:579.852.11
COBISS: 137534211 Link will open in a new window
Views: 28
Downloads: 11
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Other data

Secondary language: English
Secondary title: Introduction of electroporation for gene engineering of non-competent Bacillus subtilis strains
Secondary abstract: Wild type Bacillus subtilis strains are often characterized by lower natural competence or non-competence for DNA uptake. To study such strains, it is necessary to develop genetic manipulation tools that allow us to achieve insertion of the desired DNA segments. One of the ways to produce recombinant strains is the electroporation method, which we studied in our master's thesis. We performed experiments to optimize the method by testing different ways of preparing electrocompetent cells and electroporation parameters. Initial experiments to electroporate naturally competent B. subtilis strains 168 trpC2 and PS-216 were both successful, but electroporation of naturally noncompetent strains proved to be extremely difficult as we could not achieve the optimization of the method. To proceed with the planned experiments, we introduced the method of preparing recombinant strains by phage transduction. We succeeded in producing mutants of naturally non-competent strains, which we then used to study DNA exchange between differentially related strains during swarming on semisolid B media. Strains were labelled with an antibiotic resistance gene and a fluorescent marker. We tested DNA transfer between three combinations of non-kin strains and between one isogenic combination. Increased DNA transfer occurred between one of the tested non-kin combinations. No DNA transfer occurred in the isogenic combination. The result suggests the possibility of increased horizontal gene transfer between strains due to kin discrimination and the consequent induction of competence in otherwise naturally non-competent strains.
Secondary keywords: Bacillus subtilis;electroporation;noncompetent strains;kin discrimination;horizontal gene transfer;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za mikrobiologijo
Pages: 1 spletni vir (1 datoteka PDF (XVI, 95 str., [6] f. pril.))
ID: 17698379