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Prepoznava aminokislinskih ostankov proteina gp6 bakteriofaga GIL01 bakterije Bacillus thuringiensis ključnih za aktivacijo litičnega promotorja
Čeh, Tjaša (Author), Butala, Matej (Mentor) More about this mentor... This link opens in a new window

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Abstract
Temperatni bakteriofag (fag) GIL01 okužuje bakterijo Bacillus thuringiensis. Ob okužbi lahko v gostitelju vzpostavi litični ali lizogeni cikel. Nedavne raziskave kažejo, da so geni faga GIL01 prepisani s treh promotorjev. Litični promotor P3 nadzira prepis poznih genov za sintezo kapside in sprostitev fagov iz bakterije. Promotor P3 aktivira protein gp6, katerega gen je prepisan po poškodbi DNA s promotorja P1 faga GIL01. Protein s svojo vezavo na promotor P3 olajša vezavo RNA-polimeraze in aktivira prepis genov. Z izbranima oligonukleotidnima začetnikoma ter z DNA polimerazo Phusion smo na plazmidu uvedli točkovne mutacije v zapis za protein gp6. Sprožili smo sintezo divjega tipa proteina gp6 ali njegovih različic v sevu B. thuringiensis, ki je vključeval plazmid s poročevalskim genom lacZ pod kontrolo promotorja P3. Z beta-galaktozidaznim testom smo analizirali vpliv proteina gp6 ali njegovih različic na aktivnost P3. Z metodo površinske plazmonske resonance smo pokazali, da se protein gp6 z mutacijama v aminokislinskih ostankih gp6 Y34A in gp6 E18A veže na promotorsko regijo. V magistrskem delu smo tako dokazali, da sta točkovni mutaciji v aminokislinskih ostankih gp6 Y34A in gp6 E18A pomembni za aktivacijo promotorja P3 in s tem litičnega cikla bakteriofaga GIL01.

Language:Slovenian
Keywords:Bacillus thuringiensis, temperatni bakteriofag GIL01, gp6, litični promotor P3, odziv SOS
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2019
Publisher:[T. Čeh]
UDC:578.347:579.852.11:579.22/.26:577.2
COBISS.SI-ID:5058680 Link is opened in a new window
Views:111
Downloads:57
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Secondary language

Language:English
Title:Identification of key gp6 protein residues essential for the activation of the lytic promoter of the Bacillus thuringiensis bacteriophage GIL01
Abstract:
Temperate bacteriophage (phage) GIL01 infects insect pathogen Bacillus thuringiensis. In the host cell, the phage can trigger the lytic or the lysogenic cycle. Recent studies are showing that GIL01 genes are transcribed from three different promoters. Lytic promoter P3 is responsible for the synthesis of phage capsid and for the release of phages out of host cell. Promoter P3 is activated by the protein gp6 of which gene is transcribed from phage GIL01 promoter P1 in the DNA damage response. gp6 protein acts as an activator by interacting with the P3 promoter region, covering the -35 promoter element, which enables RNA polymerase to bind to the promoter and activates transcription of late phage genes. With set of primers and with “Phusion” DNA polymerase we introduced point mutations into the gene gp6 carried by the plasmid. We triggered the synthesis of the wild type protein gp6 or its derivates inside the strain B. thuringiensis, which carried also the plasmid with reporter gene lacZ under control of P3promoter. Using the beta-galactosidase assay, we analysed the effect of gp6 protein or its derivates on the activity of P3. We conclude that residues tyrosine 34 (gp6 Y34A) and glutamine 18 (gp6 E18A) are important for the activation of P3 promoter, thus for the activation of the lytic cycle of phage GIL01. With surface plasmon resonance we proved that protein gp6 with point mutations gp6 Y34A and gp6 E18A, bind to the P3 promoter region.

Keywords:Bacillus thuringiensis, temperate bacteriophage GIL01, gp6, lytic promoter P3, SOS response

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