Construction of Transformative System of Acetobacter xylinum

• Main Authors: Hsiu-Ling Wang, 王秀玲
• Other Authors: Yuan-Tay Shyu
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/33312122233179602556

碩士 === 國立臺灣大學 === 園藝學研究所 === 90 === Acetobacter xylinum is widely been used in food industry as it can ferment vegetables and fruits to produce the second metabolite─bacterial cellulose which is called nata in the market. However, nata is not easy for coloring, so it is essential to increase its additional value for foodstuff use. In the first part of this study, Acetobacter xylinum was transformed with pHY300PLK and pUCD2 for the finding of optimum transformation efficiency. The result showed that electroporation has the best transformation efficiency, with the conditions of 25 mF capacitance, 329 Ω ohms resistance, pulse lengths of 8.2 ms, and 2.5 kV. pHY300PLK and pUCD2 were found to have efficiency of 1.63 x 104 and 1.27 x 104 transformants / mg DNA, respectively. Cellulose binding protein gene (cbp A) and carotenoids synthesis gene were later cloned into plasmid pHY300PLK for further expression in Escherichia coli BL21 and Acetobacter xylinum CCRC14101 and protein activity been detected by Western blotting. Another plasmid pWSW8, is constructed from pUC18, an innate plasmid in Acetobacter xylinum and carotenoids synthase gene was later constructed for the increasing of stability and capability of replication, and promoting expression of foreign gene by the lac promoter within pWSW8. The result showed that carotenoids synthesis gene is not expressed significantly as predicted.