prikaz prve stranice dokumenta Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners
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master's thesis
Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2018. urn:nbn:hr:159:441360
Rupčić, Marijana
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for General Microbiology and Food Microbiology

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

Cite this document

Rupčić, M. (2018). Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:441360

Rupčić, Marijana. "Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2018. https://urn.nsk.hr/urn:nbn:hr:159:441360

Rupčić, Marijana. "Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2018. https://urn.nsk.hr/urn:nbn:hr:159:441360

Rupčić, M. (2018). 'Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 26 November 2024, https://urn.nsk.hr/urn:nbn:hr:159:441360

Rupčić M. Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2018 [cited 2024 November 26] Available at: https://urn.nsk.hr/urn:nbn:hr:159:441360

M. Rupčić, "Expression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2018. Available at: https://urn.nsk.hr/urn:nbn:hr:159:441360

Metadata
TitleExpression and characterisation of response regulator CiaR variants with His- or Flag-tags in Streptococcus pneumonia to identify CiaR interaction partners
Title (croatian)Ekspresija i karakterizacija varijanti CiaR regulatora odgovora sa His ili Flag privjescima u Streptococcus pneumoniae za identifikaciju CiaR interakcijskih partnera
AuthorMarijana Rupčić
MentorJadranka Frece (mentor)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Biochemical Engineering)
(Laboratory for General Microbiology and Food Microbiology)
Zagreb
Defense date and country2018-09-25, 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
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:159:441360
Study programmeTitle: 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 resourceText
File originBorn digital
Access conditionsAccess restricted to students and staff of home institution
Terms of useLicence In copyright icon
Created on2019-11-27 10:39:11