Karakterizacija i kultivacija knock-out sojeva bakterije Escherichia coli

master's thesis

• Title (english): Characterization and cultivation of secreting knock-out strains of Escherichia coli
• Author: Katarina Brkan
• 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: 2017-12-08, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Escherichia coli is one of the most commonly used microorganisms for production of recombinant proteins due to its numerous advantages. Many of the recombinant proteins E. coli produces are intracellular. To avoid cell disruption, ease protein yielding and purification, there have been attempts in improving extracellular protein production. One of the approaches, applied and investigated in this thesis, is using so-called leaky mutants. Due to mutations in genes encoding for proteins of the cell membrane, these mutants show an increased protein release from periplasm to nutrient medium after protein has been translocated by using natural bacterial translocation systems. Four E. coli knock-out strains have been cultivated first in flasks and then in bioreactors. Strains have been characterized regarding to their growth, protein production and secretion ability, using phytase as reporter protein.

Abstract (croatian)

Escherichia coli je jedan od najčešće korištenih mikroorganizama za proizvodnju rekombinantnih proteina od kojih su mnogi intracelularni. Kako bi se izbjeglo mehaničko razbijanje stanica, olakšala izolacija i pročišćavanje proteina pokušava se unaprijediti proces ekstracelularne proizvodnje proteina. Jedan od pristupa, primijenjen i istražen u ovom radu je korišenje takozvanih leaky mutanata. Zbog mutacija u genima koji kodiraju za proteine stanične stijenke, ovi mutanti bi trebali pokazivati povećanu sposobnost izlučivanja proteina iz periplazme u hranjivu podlogu nakon translokacije proteina prirodno prisutnim bakterijskim translokacijskim mehanizmima. Četiri knock-out soja bakterije E. coli su kultivirani najprije u tikvicama a zatim i u bioreaktorima. Kultivirani sojevi su karakterizirani obzirom na rast, koncentraciju proizvedenog proteina i sposobnost sekrecije koristeći fitazu kao reporter protein.

• Keywords: E. coli; cell wall; secretion; leaky mutants; phytase
• Keywords (english): E. coli; stanična stijenka; sekrecija proteina; leaky mutanti; fitaza
• Language: english
• URN:NBN: urn:nbn:hr:159:750958
• 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: 2019-11-21 14:16:11