Folate auxotrophic Saccharomyces cerevisiae: construction and preliminary characterization
Examensarbete för masterexamen
Biotechnology (MPBIO), MSc
Folate, vitaminB9, is an essential methyl donor involving in synthesis of DNA, protein, and phospholipids as well as participating in one-carbon metabolism. In all organisms, folate is required for methionine homeostasis entailing in methylation reactions including those of epigenetic modiﬁcations. With folate deﬁciency being one of the most common nutritional deﬁciency experiencing worldwide, its effects especially on gene regulation are worth exploring. In this study, Saccharomyces cerevisiae was chosen to construct a model organism in order to investigate the effect of folate deﬁciency on epigenetic modiﬁcations in term of oxidative stress resistance. As S. cerevisiae is able to synthesize folate de novo, the folate biosynthesis pathway has to be disrupted to enable folate deﬁcient environment. Subsequent to difﬁculty in achieving the mutants, UV mutagenesis was ﬁrst used to select for the strain that was able to utilize external folate in the form of folinic acid more efﬁciently. Then, FOL3 gene locating within folate biosynthesis pathway, was disrupted via PCR-mediated gene disruption to achieve the folate auxotrophic strain. Following the strain construction, growth characteristics were analyzed in microscale. Compared to the prototrophic strain, folate auxotroph supplemented with 100 µg/mL folinic acid in minimal media exhibited some growth limitation. However, when exposed to oxidative stress, no difference was observed between folate auxotroph and prototroph after UV mutagenesis. This work only serves as a starting point and further investigation has to be done before this strain can be apply as a model to study epigenetics.
Biokemi och molekylärbiologi , Mikrobiologi , Livsmedelsvetenskap , Livsvetenskaper , Biochemistry and Molecular Biology , Microbiology , Food Science , Life Science