Konstrukcija plazmidnog vektora za transformaciju kvasca Pichia pastoris

undergraduate thesis

• Title (english): Construction of shuttle vector for the transformation of yeast Pichia pastoris
• Author: Josip Rešetar
• Mentor: Renata Teparić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2018-07-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Pichia pastoris često je korišteni mikroorganizam u biotehnološkoj proizvodnji heterolognih proteina. Zbog mogućnosti rasta do visokih staničnih gustoća važna je mogućnost njegove primjene u dobivanju kemikalija na ekološki prihvatljiv i ekonomski isplativ način. U ovom radu konstruiran je plazmidni vektor pogodan za transformaciju kvasca Pichia pastoris koji nosi gen za rekombinantni protein Ccw12XR iz kvasca Saccharomyces cerevisiae. Rekombinantni gen se sastoji od dijela koji kodira za enzim ksiloza reduktazu (XR) i dijela koji kodira za protein Ccw12 stanične stijenke kvasca S. cerevisiae. Rekombinantni gen umnožen je PCR metodom i ugrađen TA ligacijom u plazmidni vektor pGEM®-T Easy. Plazmid je umnožen u kompetentnim stanicama E. coli, nakon čega je iz plazmida izrezan rekombinantni gen CCW12XR korištenjem SapI restrikcijskog enzima. Naposljetku je CCW12XR gen ligiran s lineariziranim plazmidom pBSY3Z. Uspješnost ligacije je, nakon umnažanja plazmidnog vektora u kompetentnim stanicama E. coli, provjerena restrikcijom pomoću odabranih restrikcijskih enzima i elektroforezom na agaroznom gelu.

Abstract (english)

Pichia pastoris is a commonly used host in the biotechnological production of heterologous proteins. Since it can grow to very high cell densities, Pichia pastoris is applicable in the eco-friendly and economical production of chemicals. This thesis describes the construction of the shuttle vector, suitable for transformation of Pichia pastoris carrying the gene for the Ccw12XR recombinant protein from Saccharomyces cerevisiae. The recombinant gene encodes both the xylose reductase (XR) and the Ccw12 cell wall protein of S. cerevisiae. The recombinant gene is amplified using PCR and ligated into pGEM®-T Easy. The plasmid is replicated in competent Escherichia coli cells, following which the recombinant CCW12XR gene is restricted from the constructed plasmid using SapI. The isolated CCW12XR gene is then ligated with the linearized pBSY3Z plasmid. Finally, following the replication of the shuttle vector in component E. coli cells, the ligation efficiency is verified via restriction analysis and DNA electrophoresis.

• Keywords: CCW12XR; ksiloza reduktaza; Pichia pastoris; plazmidni vektor
• Keywords (english): CCW12XR; Pichia pastoris; shuttle vector; xylose reductase
• Language: croatian
• URN:NBN: urn:nbn:hr:159:999781
• Study programme: Title: Nutrition; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) nutricionizma (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) nutricionizma)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-07-19 09:20:28