Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners

master's thesis

• Title (croatian): Ekspresija i karakterizacija varijanti CiaR regulatora odgovora sa His ili Flag privjescima u Streptococcus pneumoniae za identifikaciju CiaR interakcijskih partnera
• Author: Marijana Rupčić
• Mentor: Jadranka Frece (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for General Microbiology and Food Microbiology); Zagreb
• Defense date and country: 2018-09-25, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

In CiaRH two component regulatory system in Streptococcus pneumoniae, under certain conditions, CiaH histidine kinase is not needed for phosphorylation of CiaR response regulator. Acetyl phosphate AcP is most likely the alternative donor of phosphate for CiaR when CiaH is missing. There are some indications that AckA acetate kinase acts negatively on CiaR phosphorylation via AcP. The aim of this work is to detect interactions between CiaR and AckA in Streptococcus pneumoniae (in vivo). For easier purification, two CiaR tagged variants (with His and Flag tag) were expressed. CiaR without tag was used as a control. Activity of CiaR variants was determined through measurement of β-galactosidase activity. After isolation and purification of CiaR tagged variants, fractions were analysed with Western blot for the presence of AckA, possible interaction partner. Cross- linking was also performed and CiaR-His variant was purified. Fraction was analysed with Western blot for the presence of AckA. AckA was not detected in any fractions with purified CiaR. Interaction of CiaR and AckA has not been detected in vivo so far by the applied methods.

Abstract (english)

U CiaRH dvokomponentnom sistemu bakterije Streptococcus pneumoniae, u određenim uvjetima, CiaH histidin kinaza nije potrebna za fosforilaciju CiaR regulatora odgovora. Acetil fosfat je najvjerovatnije alternativni donor fosfata za regulator odgovora kada nedostaje CiaH. Pretpostavlja se da AckA acetat kinaza djeluje negativno na tu fosforilaciju, vrlo vjerovatno direktnim stupanjem u interakcije sa CiaR. Cilj ovog rada je detektirati interakcije između CiaR i AckA u Streptococcus pneumoniae (in vivo). U ovom radu su eksprimirane dvije varijante CiaR sa različitim proteinskim privjescima (His i Flag) zbog lakšeg pročišćavanja. CiaR bez privjeska je korišten kao kontrolna varijanta. CiaR varijantama je ispitana aktivnost preko mjerenja aktivnosti β-galaktozidaze. Nakon izolacije i pročišćavanja CiaR varijanti sa privjescima, u frakcijama je ispitana prisutnost AckA, mogućeg interakcijskog partnera, pomoću Western blota. Također je proveden i cross-linking nakon kojeg je pročišćena CiaR-His varijanta. Frakcija je analizirana na prisutnost AckA pomoću Western blota. Ni u jednoj od frakcija sa pročišćenim CiaR varijantama nije detektirana AckA. Interakcije između CiaR i AckA nisu detektirane (in vivo) sa primjenjenim metodama.

• Keywords: CiaRH two component system; Streptococcus pneumoniae; CiaR response regulator; AckA acetate kinase
• Keywords (croatian): CiaRH; Streptococcus pneumoniae; CiaR regulator odgovora; AckA acetat kinaza
• Language: english
• URN:NBN: urn:nbn:hr:159:441360
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2019-11-27 10:39:11