Analiza lastnosti proteina gp7 bakteriofaga GIL01

Zupančič, Špela (author), Butala, Matej (mentor)

Proteina RecA in LexA sta ključna proteina tako imenovanega odziva SOS, ki omogoča ohranitev integritete in strukture genoma bakterij, ki rastejo v stresnih, genotoksičnih razmerah. V tem odzivu pa se lahko sproži tudi prehod nekaterih bakteriofagov iz lizogenega v litičen cikel. Bakteriofag GIL01, ki okužuje bakterijo Bacillus thuringiensis, za vzpostavitev lizogenega cikla izkorišča gostiteljev protein LexA. Protein LexA se veže na tarčna mesta v promotorski regiji P1 bakteriofaga GIL01, to vezavo pa dodatno stabilizira protein gp7 tega bakteriofaga. Kompleks LexA-gp7 prepreči bakteriofagu prehod v litični cikel. V magistrski nalogi smo preverili, ali lahko protein gp7 prosto prehaja iz gojišča v bakterijo. Z afinitetno kromatografijo smo očistili protein gp7 in analogni protein DdrR iz bakterije Acinetobacter baumannii. Nadalje smo očistili protein gp7, katerega smo sklopili s signalnim zaporedjem YKKSNNPFSD, ki naj bi omogočilo vnos proteinov v bakterijo Bacillus subtilis. Za odstranitev afinitetne značke na amino-terminalnem koncu proteinov smo uporabili encim enterokinaza. Zasnovali smo test prehoda proteinov gp7 v Bacillus thuringiensis serovar israelensis, ki temelji na analizi promotorske aktivnosti P1 z β-galaktozidaznimi testi. Dokazali smo, da je protein gp7 stabilen v izrabljenem gojišču bakterije B. thuringiensis. Rezultati nakazujejo, da gp7 ali gp7 s signalno sekvenco ne prehajata prosto v bakterijo.

• Language: Slovenian
• Keywords: protein gp7, signalne sekvence, Bacillus thuringiensis, inhibicija odziva SOS
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2021
• PID: 20.500.12556/RUL-124895
• COBISS.SI-ID: 55208451
• Publication date in RUL: 25.02.2021

Secondary language

• Language: English
• Title: The analysis of bacteriophage GIL01 gp7 protein characteristics

Abstract

The proteins RecA and LexA are important factors in the SOS response that enables bacterial genome integritiy under stressful genotoxic conditions. SOS response also induces bacteriophages transition from the lysogenic to the lytic cycle. The bacteriophage GIL01 infects the bacterium Bacillus thuringiensis and requires host`s LexA repressor to establish and maintain the lysogenic cycle. LexA binds to specific sites in the P1 promoter region of the GIL01 bacteriophage. The LexA protein can bind gp7 which additionally enhances the binding of LexA to DNA and prevents the bacteriophage from switching to the lytic cycle. We tested whether gp7 could freely enter into the bacterium. We purified gp7 and its analog from the bacterium Acinetobacter baumannii, the protein DdrR with affinity chromatography and also gp7 with the signal sequence YKKSNNPFSD, which presumably allows the fusion protein to enter into the bacterium Bacillus subtilis. To remove the histidine affinity tag at the amino-terminal part of the protein, we used enterokinase. We designed the gp7 protein transformation in Bacillus thuringiensis serovar israelensis based on analyzes of the activity of the P1 promoter with β-galactosidase assays. We proved that gp7 is stable in the spent culture medium. The results show that gp7 or its analog carrying the signal sequence cannot freely migrate into the bacterium.

• Keywords: gp7 protein, signal sequences, Bacillus thuringiensis, inhibition of SOS response