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題名 | 鹼性枯草桿菌YaB電轉形最適條件之探討=Optimum Conditions for the Transformation of Alkalophilic Bacillus YaB Electroporation |
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作者 | 葉娟美; 蔡英傑; | 書刊名 | 中國農業化學會誌 |
卷期 | 37:3 1999.09[民88.09] |
頁次 | 頁348-359 |
分類號 | 369.4 |
關鍵詞 | 電轉形; 鹼性枯草桿菌; Electroporation; Alkalophilic Bacillus YaB; |
語文 | 中文(Chinese) |
中文摘要 | 鹼性枯草桿菌YaB可分泌鹼性彈性蛋白□。此酵素被認為是甚具潛力的肉品軟化酵素,其基因已被選殖出,但在宿主細胞之表現量遠不及原生產株。為求在原生產株之染色體上進行改造以提高其產量,有必要先建立原始酵素生產菌之轉形法。鹼性枯草桿菌之DNA轉形法甚少見諸報告,近年電轉形法由於操作簡便快捷,發展相當迅速,受到極大重視。本報告對鹼性枯草桿菌電轉形法各項相關因子作一深入探討。前培養條件以添加甘胺酸之培養液,震盪培養至OD□約為0.5左右為最佳。電轉形緩衝液則以20% PEG之SMP緩衝液轉形效率較高。菌液與質體之比則為菌液100Μl;0.8-1.0μg質體,菌體濃度為10□CFU/mL。電轉形之施壓以電容25μF、電壓3.0 kV/cm、電阻400Ω(相當於9.2msec施壓時間)為最佳條件。最佳電轉形效率可達10□CFU/μg DNA。 |
英文摘要 | Alkalophilic Bacillus YaB can produce alkaline elastase, which is considered to be a potent meat tenderizer. The structural gene ale has been cloned, sequenced and expressed in B. subtilis, but the expression level was much lower than in the wild strain. To further improve the expression level in the wild strain or construct mutants from the wild strain, an efficient transformation of Alkalophilic Bacillus YaB was critical. This study, and electroporation-mediated transformation was optimized for the strain YaB. The optimal pre-culture condition was shaking-cultured in a LB/NaCL/glycine medium until the OD600 reached about 0.5. The cells are washed and suspended in SMP buffer containing 20% PEG as the electroporation buffer, and the cell to plasmid was found to be 100μL cells: 0.8~1.0 μg DNA in each cuvette Electroporation was performed under a capacitance of 25μF、a field strength of 3.0kV/cm and 400Ω (about 9.2 msec duration time) using a Bio-Rad gene pulser. The cells were then regenerated in LB/NaCL broth under gentle shaking for another 60 minutes. Using the optimal conditions obtained, a transformation efficiency of 104 transformants per μg DNA could be achieved. |
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