Evrimsel Mühendislik Yöntemi İle Demire Dirençli Saccharomyces Cerevisiae’nın Biyomimetik Amaçlı Eldesi
Evrimsel Mühendislik Yöntemi İle Demire Dirençli Saccharomyces Cerevisiae’nın Biyomimetik Amaçlı Eldesi
Dosyalar
Tarih
2010-07-13
Yazarlar
Balaban, Berrak Gülçin
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Fen Bilimleri Enstitüsü
Institute of Science and Technology
Institute of Science and Technology
Özet
Bu çalışmada, bir tersine metabolik mühendisliği stratejisi olan evrimsel mühendislik uygulanarak, demire dirençli Saccharomyces cerevisiae mayaları elde edilmiştir. Başlangıç kültürüne, kimyasal mutajen olan etil metan sülfonat uygulanarak, genetik çeşitliliği arttırılmıştır. Demire dirençli mutantların seçimi, hem hücrelere 5 ve 30mM FeCl2 aralığında sürekli stres uygulanarak, hem de eksponansiyel büyüme fazında, 5 ve 180mM FeCl2 aralığında ani stres, uygulanarak elde edilmiştir. Sürekli stres seçilim yöntemi ile 30mM FeCl2’e kadar hayatta kalabilen 15 nesil ve ani stres seçilim yöntemi ile 180mM ani FeCl2 stresine dirençli 25 nesil elde edilmiştir. Son mutant nesillerden, mutant bireyler seçilmiş ve bunların demir direnç seviyeleri ile diğer metallere ve diğer stres çeşitlerine karşı çapraz dirençleri belirlenmiştir. Demire dirençli mutantların kobalt ve nikel streslerine de çapraz direnç gösterdikleri belirlenmiştir. Sonuçlar demir, kobalt ve nikel stres direncinin moleküler mekanizmalarının mayada benzer olabileceğine işaret etmektedir.
In this study, evolutionary engineering, an inverse metabolic engineering strategy, was applied to improve iron resistance of Saccharomyces cerevisiae. Chemical mutagenesis by ethyl methane sulfonate was performed to the initial culture to increase genetic diversity. Selection for iron resistant mutants was applied in batch cultures, under continuously applied stress conditions ranging between 5 and 30mM FeCl2, as well as under pulse stress conditions between 5 and 180mM FeCl2 that were applied to the cells for 90 minutes during their exponential phase of growth. From continuous stress selection; 15 generations were obtained that could survive up to 30mM FeCl2 and the pulse selection yielded 25 generations which resisted up to 180mM FeCl2 applied as a pulse stress. Individual mutants were selected from the final mutant populations and their iron resistance levels, as well as their cross-resistance to other metals and other stress types were determined and discussed. It was found that iron-resistant mutants also had cross-resistance against cobalt and nickel stresses. The results imply that the molecular mechanisms of iron, cobalt and nickel stress resistance may be similar in yeast.
In this study, evolutionary engineering, an inverse metabolic engineering strategy, was applied to improve iron resistance of Saccharomyces cerevisiae. Chemical mutagenesis by ethyl methane sulfonate was performed to the initial culture to increase genetic diversity. Selection for iron resistant mutants was applied in batch cultures, under continuously applied stress conditions ranging between 5 and 30mM FeCl2, as well as under pulse stress conditions between 5 and 180mM FeCl2 that were applied to the cells for 90 minutes during their exponential phase of growth. From continuous stress selection; 15 generations were obtained that could survive up to 30mM FeCl2 and the pulse selection yielded 25 generations which resisted up to 180mM FeCl2 applied as a pulse stress. Individual mutants were selected from the final mutant populations and their iron resistance levels, as well as their cross-resistance to other metals and other stress types were determined and discussed. It was found that iron-resistant mutants also had cross-resistance against cobalt and nickel stresses. The results imply that the molecular mechanisms of iron, cobalt and nickel stress resistance may be similar in yeast.
Açıklama
Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2010
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2010
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2010
Anahtar kelimeler
Saccharomyces cerevisiae,
demir direnci,
evrimsel mühendislik,
Sacharomyces cerevisiae,
iron resistance,
evolutionary engineering