臺灣博碩士論文加值系統

本實驗的目的除了建立Streptomyces的轉型系統外，並在Streptomyces中找出具誘導性且為較強勢的啟動子，希望藉此建立一個經濟且高效率的表現系統來大量表現異源性基因，除此之外，也希望將此表現系統應用於clavulanic acid的生合成。將 S. lividans TK64 培養 36 小時，當菌體量達達 0.7 ml/ml PCV (Packed cell volume)時，此種菌體可產生最多量的原生質體，濃度達 2.3×1010 CFU/ml。進行轉形的 DNA 濃度愈高，其轉形效率愈好，而且利用 PEG8000 最適合S. lividans TK64原生質體轉形作用，最高轉形效率可達 5.1×104 transformants/μg DNA。本研究也將 S. lividans TK24的 xlnA 及 xlnB 基因之啟動子區 (promoter region) 選殖於報導基因 (Pseudomonas putida 的 xylE 基因) 的 5 端，在轉形至 E. coli ，利用 1% xylan 或 1mM IPTG 誘導下，發現包含xlnA 基因上游區 600bp 啟動子片段的轉形株 E. coli (pUC-XA) 表現最好，catechol dioxygenase 之比活性達 75U/mg。xlnB 基因之啟動子區，則是包含 xlnB 基因上游區 650bp 啟動子片段帶動xylE基因的表現最佳，E. coli (pUC-XB) 之catechol dioxygenase 比活性達 650U/mg。將這些構築轉形入 S. lividans TK64，於 NMMP broth 內以 1% xylan 誘導，S. lividans (pIJ-XA) 之 xylE 基因表現最好，catechol dioxygease 比活性達 750mU/mg，而 xlnB 啟動子中則以S. lividans (pIJ-XBS) 之xylE 基因的表現最好， catechol dioxygenase 比活性達 200mU/mg。

中文摘要…………………………………………………………………1
前 言………………………………………………………………….3
材料與方法……………………………………………………………….11
一、 藥品……………………………………………………………….…11
二、 菌株、質體及培養基…………………………………………….…11
三、 E. coli 質體 DNA 的抽取與DNA片段的回收……………….…13
四、 S. lividans TK24 染色體 DNA 的抽取…………………………...14
五、 DNA 的定序與洋菜膠電泳分析…………………………………..14
六、 DNA 純度之鑑定及定量分析……………………………………..15
七、 引子的設計………………………………………………………….15
八、 聚合酉每鍊鎖反應…………………………………………………….16
九、 DNA 黏接反應……………………………………………………...17
十、 質體轉形作用………………………………………………….…….17
十一、 蛋白質之定量、電泳……………………………………………17
十二、 S. lividans TK64 轉形系統的建立……………………………..18
1.原生質體的製備………………………………………………..18
2.原生質體的轉形作用…………………………………………..19
3.小量製備放線菌之質體………………………………………..20
十三、Strptomyces lividans TK64 利用原生質體法轉形最佳條件之探討
1. 不同生長階段的菌絲對原生質體生成數的影響……………21
2. DNA 濃度對轉形株數目的影響…………………………..…21
3. 不同分子量的 PEG 對轉形效率的影響…………………….22
十四、含 xlnA 或 xln B 基因啟動子之質體之構築……………………22
十五、xln 基因啟動子在 E. coil的表現能力……………………………24
(1) 平板分析法…………………………………………………….24
(2) 酵素活性分析法……………………………………………….24.
十六、 xln 基因啟動子基因選殖入 S. lividans 載體 pIJ702……….25
十七、 xln 基因起動子在 S. lividans TK64 之表現能力…………...26
十八、 Xylan 濃度對 xln 基因啟動子活性的影響………………….27
十九、Clavulanic acid 生合成基因串之組裝…………………………27
(1) pUC19ST表現載體之構築……………………………….27
(2) 將 clavulanic acid 基因選殖入 pUC19ST………………..28
結 果
一、 S. lividans TK64 原生質體轉法形最佳條件之探討……………29
(1) 不同生長階段的菌絲對原生質體生成的影響…………….29
(2) DNA 濃度對轉形的影響……………………………………29
(3) PEG 對轉形效率的影響…………………………………….29
二、 xln 基因啟動子在 E. coil 中之表現能力……………………….30
三、 xln 基因啟動子在S. lividans TK64中之表現能力……………...31
四、基質濃度對 xln 基因啟動活性的影響……………………………32
五、Clavulanic acid 生合成基因串之組裝……………………………..33
討 論
一、 xln 基因啟動子在E. coil之功能性………………………………35
二、 xln 基因啟動子的長度與基因在 S. lividans TK64 表現之關係..37
三、Streptomyces spp.基因啟動子強度之比較………………………….39
四、利用 Streptomyces 表現系統生產 clavulanic acid………………..40
圖表……………………………………………………………………….42
參考文獻………………………………………………………………….74

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