Cultivation and characterization of E. coli protein – secretion strains

master's thesis

• Title (english): Uzgoj i karakterizacija E. coli protein – sekrecijskih sojeva
• Author: Laura Jurić
• 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: 2018-07-13, 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. The vast majority of proteins are produced intracellular, either in cytoplasm or periplasm, so that obtaining a biologically active proteins represents a challenge. With extracellular production of proteins, would be avoided cell disruption, ease protein yielding and purification. 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. Two E. coli knock-out strains have been cultivated first in flasks (without and with pAppA plasmid) 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. Veliku većinu proteina proizvodi intracelularno, bilo u citoplazmi ili u periplazmi, tako da dobivanje biološki aktivnih proteina predstavlja izazov. Ekstracelularnom proizvodnjom proteina izbjeglo bi se mehaničko razbijanje stanica, olakšala izolacija i pročišćavanje proteina. Jedan od pristupa, primjenjen i istražen u ovome radu, je upotreba 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. Dva knock-out soja bakterije E. coli su kultivirani najprije u tikvicama (bez i s pAppA plazmidom) 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 (croatian): E. coli; stanična stijenka; sekrecija proteina; leaky mutanti; fitaza
• Language: english
• URN:NBN: urn:nbn:hr:159:400382
• 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 12:54:57