Development of a drug-free selection system using 2A-mediated cleavage in methylotrophic yeast Pichia pastoris
Date Issued
2011
Date
2011
Author(s)
Chiang, I-Lun
Abstract
Antibiotic and herbicide resistance genes have been commonly used as selectable markers in genetically modified microorganisms and transgenic plants. However, problems with the widespread use of transgenes arise when they are dispersed throughout an ecosystem through either pollen or horizontal gene transfer by soil microorganisms. To partially alleviate this problem, we sought to develop a drug-free selection system in the methylotrophic yeast Pichia pastoris. To do so, we constructed a vector for transgenic protein expression that harnesses both the reporter gene egfp and 2A-mediated cleavage. As a proof of principle, a bicistronic expression vector was constructed with gus and egfp linked by a 2A peptide cloned downstream of the AOX1 promoter. In our first construct, we included an antibiotic resistance gene and screened transformants by antibiotics. Extracellular supernatants were analyzed by protein electrophoresis and GUS assay. EGFP expression was analyzed by fluorescent microscopy and fluorescent-activated cell sorting (FACS). Once it was shown that our 2A-mediated cleavage was successful and that both GUS and EGFP were properly expressed, we constructed the same vector, but without the antibiotic resistance gene. After transformation of the antibiotic marker-free plasmid into P. pastoris, we screened for transformants by EGFP expression with FACS. The transformants selected by FACS cell sorter demonstrate that GUS activity and EGFP expression level has positive correlation, which proves the hypothesis of equimolar expression of protein upstream and downstream of 2A peptide in bicistronic vector.
Subjects
drug-free selection system
2A-mediated cleavage
Pichia pastoris
EGFP
Type
thesis
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