Karakterizacija bakteriofaga, zapisanega na plazmidu pBtic235 bakterije Bacillus thuringiensis

Miklavčič, Urša (author), Butala, Matej (mentor)

Bakterija Bacillus thuringiensis subsp. israelensis vsebuje poleg kromosoma še številne molekule plazmidne DNA, ki so sposobne samostojnega podvojevanja. Eden izmed plazmidov je linearen in vsebuje gene bakteriofaga GIL01, drugi, krožni plazmid pBtic235 vsebuje gene slabše okarakteriziranega bakteriofaga. V magistrski nalogi smo se osredotočili na vpliv proteinov LexA bakterije B. thuringiensis in gp7 bakteriofaga GIL01 na prehod bakteriofaga, zapisanega na plazmidu pBtic235 iz lizogenega v litični življenjski cikel. S prenosom western smo želeli preveriti aktivnost protiteles v serumih proti proteinom gp6, gp7 bakteriofaga GIL01 in proti proteinu LexA bakterije B. thuringiensis. Slednja protitelesa bi nam služila pri analizi vpliva proteinov gp6, gp7 ter LexA na uravnavanje genetskega stikala bakteriofaga, zapisanega na pBtic235. Validacija protiteles potrjuje specifično aktivnost protiteles proti proteinu LexA. Analizirali smo pomen proteina gp7 na iniciacijo litičnega cikla bakteriofaga, zapisanega na pBtic235, s spremljanjem rastnih krivulj različnih sevov po indukciji z mitomicinom C. Potrdili smo, da poškodba DNA sproži litični cikel bakteriofaga, zapisanega na plazmidu pBtic235. Poleg tega naši rezultati nakazujejo, da protein gp7 bakteriofaga GIL01 vpliva na časovno uravnano sprožitev litičnega cikla bakteriofaga, kodiranega na pBtic235. Testirali smo tudi indukcijo bakteriofaga s plazmida pBtic235 v sevu bakterije z okvarjenim LexA proteinom, ki se ne more samoinaktivirati. Rezultati nakazujejo, da je protein LexA pomemben tudi za uravnavanje litičnega/lizogenega cikla bakteriofaga, zapisanega na pBtic235.

• Language: Slovenian
• Keywords: Bacillus thuringiensis, plazmid pBtic235, bakteriofag, LexA, gp7, preklop iz lizogenega v litični cikel
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2021
• PID: 20.500.12556/RUL-125560
• COBISS.SI-ID: 59729411
• Publication date in RUL: 25.03.2021

Secondary language

• Language: English
• Title: Characterisation of bacteriophage encoded on the Bacillus thuringiensis plasmid pBtic235

Abstract

Bacterium Bacillus thuringiensis subsp. israelensis contains, in addition to the chromosome, several plasmid DNA molecules capable of self-replication. One of the plasmids is linear and carries bacteriophage GIL01 and on the circular plasmid pBtic235, there are genes of another less well-characterized bacteriophage. In the master's thesis we focused on influence of proteins LexA of B. thuringiensis and gp7 from bacteriophage GIL01 on the transition of life cycles of bacteriophage encoded on pBtic235. With western blot we tested the activity of antibodies against proteins gp6, gp7 of bacteriophage GIL01 and against protein LexA from B. thuringiensis. Later antibodies would serve us in the analysis of the influence gp6, gp7 and LexA proteins have on the regulation of genetic switch of life cycle of bacteriophage pBtic235. Validation of antibodies confirmed the activity of antibodies against protein LexA. We analysed the significance of gp7 protein on the initiation of the lytic cycle of pBtic235 phage, by monitoring the growth of different strains after induction by mitomycin C. We confirmed that DNA damage triggers the lytic cycle of phage encoded on plasmid pBtic235. Our results also suggest that the gp7 protein of the bacteriophage GIL01 influences the time-regulated initiation of the lytic cycle of the bacteriophage encoded by pBtic235. Phage induction from plasmid pBtic235 in a bacterial strain with defective LexA protein that cannot self-cleave was also tested. The results suggest that the LexA protein is also important for the regulation of the lytic/lysogenic cycle of phage carried by pBtic235.

• Keywords: Bacillus thuringiensis, pBtic235 plasmid, phage, LexA, gp7, lysogenic/lytic switch