Development of methods for genomic insertion of genes in microorganisms

• Main Authors: Ming-zhi Li, 李名智
• Other Authors: Yun-peng Zhao
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/91185142131916224249

碩士 === 逢甲大學 === 化學工程學所 === 98 === The methods for genomic insertion of genes into Escherichia coli and baker yeast, respectively, were developed in this study. For E. coli, the composite transposon Tn5 was constructed to carry target genes along with an antibiotic determinant flanked by two loxP sites. By transposition, the target genes were randomly integrated into E. coli genome aimed at enhancing the activity of key reaction steps in metabolic pathway. Later removal of marker by the act of Cre allowed the cycling integration of the target genes carried on the transposon. As a result, the pathway flux could be redirected to the desired product. For baker yeast, a markerless method for genomic insertion of genes was developed. This was based on the construction of a DNA cassette consisting of the gene to be inserted and the loxP site-flanked antibiotic maker gene. By this method, the gene encoding XR and XDH from Pichia stipitis was inserted into yeast genome. In addition, a method for insertion of the artificial promoter upstream of genomic genes was also developed. Consequently, endogenous gene XK of yeast was overexpressed.