Improvement of plasmid stability in E. coli for industrial production

master's thesis

• Title (english): Povećanje stabilnosti plazmida u E. coli za industrijsku proizvodnju
• Author: Lukrecija Vlahović
• Mentor: Božidar Šantek (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2018-10-26, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

E. coli is most common host for recombinant protein production. Majority of the expression systems based on it depend on attendance of at least one plasmid that needs to be segregated into dividing cells during growth. Segregational plasmid stability is one of the major issues of industrial scale production of recombinant proteins during which conventional method of adding antibiotics should be excluded. One of the alternatives is to use auxotrophic mutants transformed with plasmid that contains gene of interest and gene that complements the mutation. In this thesis mutant strain of E coli with deleted gene for triosephosphate isomerase, transformed with plasmid which complements mutation and has resistance to antibiotic, is used. Its growth and segregational stability of the plasmid is compared to the transformed parent strain, cultivated in medium with and without antibiotic. In addition, new auxotrophic strain, with lower probability of creating revertants is created.

Abstract (croatian)

E. coli je najčešći domaćin za proizvodnju rekombinantnih proteina. Većina ekspresijskih sistema baziranih na njoj ovisi o pristutnosti barem jednog plazmida koji se treba pravilno rasporediti u stanice kćeri tijekom diobe. Segregacijska stabilnost plazmida je jedan od najvećih problema tijekom industrijske proizvodnje rekombinantnih proteina tijekom koje je potrebno izbjeći uobičajenu metodu dodatka antibiotika kulturi, a jedan od alternativnih načina je upotreba auksotrofnih mutanata transformiranih plazmidom koji uz gen od interesa sadrži gen koji komplementira mutaciju soja. U ovom radu korišten je mutiran soj E. coli s deletiranim genom za trioza fosfat izomerazu bez kojeg bakterija ne može rasti na glicerolu. Soj je transfomiran plazmidom koji komplementira mutaciju te ima rezistenciju na antibiotik. Rast mutiranog soja i segregacijska stabilnost plazmida uspoređeni su sa roditeljskim, ne mutiranim sojem, transformiranim istim plazmidom, te uzgajanim u mediju sa i bez antibiotika. Uz to, kreiran je novi auksotrofni soj, sa smanjenom mogućnošću pojave revertanata

• Keywords: E. coli; segregational stability; triosephosphate isomerase
• Keywords (croatian): E. coli; segregacijska stabilnost; trioza fosfat izomeraza
• Language: english
• URN:NBN: urn:nbn:hr:159:234166
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-11-26 12:47:48