臺灣博碩士論文加值系統

藍藻蛋白 (Cyanophycin) 又稱藻青素，由天門冬胺酸 (L-aspartic acid, Asp) 與精胺酸 (L-arginine, Arg) 以等莫耳數所組成，其分子量介於 25~100 kDa；於工業上可被用來製造聚天門冬胺酸，並廣泛用於生物醫學及藥品製造。
本實驗室先前將源自於 Synechocystis sp. PCC6803 藍藻蛋白合成酶基因 (cphA)，選殖入 pRham N-His SUMO Kan Vector 並轉形至 Escherichia coli NEB-turbo 中表現，在 0.1% rhamnose 下誘導24 小時，提高藍藻蛋白產量。然而，在 TB 培養基中此重組菌株藍藻蛋白產量偏低，因此實驗取三種含有 cphA 基因的載體 (pRham-cphA、pET-21b-cphA、pET-21b-His-cphA) 轉形至CleanColi BL21(DE3)、CleanColi BL21(DE3)-CodonPlus 和 E. Coli BL21(DE3)-CodonPlus-RIL 宿主菌株中探討藍藻蛋白產量，其中三個載體在去內毒素大腸桿菌中皆表現不佳，且實驗室先前已知 pET-21b-cphA 和 pET-21b-His-cphA 在 E. coli BL21(DE3)-CodonPlus-RIL 中藍藻蛋白產量不高，故將 pRham N-His-cphA plasmid 轉形至 E. coli BL21(DE3)-CodonPlus-RIL中，發現其藍藻蛋白產量較高，且在添加 0.1% rhamnose 誘導物質並於 20℃ 下 100 rpm 培養 24 小時，可獲得較佳藍藻蛋白產量。
利用單ㄧ因子試驗分析，選定 tryptone、yeast extract 與 potassium phosphates 進行培養基最適化探討，再由反應曲面法取得統計分析，藉由數據得知 tryptone 在 TB 培養基中不可或缺，yeast extract 在 TB 培養基中含量提升至 1.37 倍，藍藻蛋白產量最高，potassium phosphates 則與藍藻蛋白產量呈負相關，以電腦軟體計算得到最適化 TB 培養基配方為 2.4% tryptone、3.28% yeast extract、0.4% glycerol、50 mM aspartic acid、50 mM arginine，添加 0.2% rhamnose 於 20℃ 下 100 rpm 震盪培養 24小時可得到最佳藍藻蛋白產量。

關鍵詞：藍藻蛋白、培養基最適化、反應曲面法。

Cyanophycin is a product from cyanobacteria, which is also called blue-green algae. Cyanophycin is composed by L-aspartic acid and L-arginine in impartial moles. The molecular weight of cyanophycin is around 25-100 kDa. It can be used to produce polyaspartic acids in industry, and can be widely applied in the biomedical and pharmaceutical manufacturing. Our laboratory had previously cloned the gene of cyanophycin synthetase into pRham N-His SUMO Kan Vector, expressed in E. coli-NEB-turbo to produce cyanophycin, and found that the optimal induction was carried out with 0.1% of rhamnose for 24 hours at 20℃ after the OD600 reaching 0.4. However, recombinant E. coli NEB-turbo carrying pRham-cphA vector grew poorly in TB medium, and has a low yield of cyanophycin. Thence, three vectors carrying cphA (pRham-cphA, pET-21b-cphA, and pET-21b-His-cphA) were respectively transferred into CleanColi BL21(DE3), CleanColi BL21(DE3)-CodonPlus, and E. Coli BL21(DE3)-CodonPlus-RIL cells, respectively. We found that these three vectors in CleanColi BL21(DE3) and CleanColi BL21(DE3)-CodonPlus all have resulted low yields of cyanophycin. Our laboratory also had previously transferred pET-21b-cphA and pET-21b-His-cphA, respectively, into E. coli BL21(DE3)-CodonPlus-RIL, and found that the yields of cyanophycin weren’t very high. When pRham N-His-cphA was transferred into E. coli BL21(DE3)-CodonPlus-RIL, a high yield of cyanophycin can be obtained by induction with 0.1% of rhamnose under 100 rpm, 20℃ for 24 hours. Tryprone, yeast extract and potassium phosphates were choosen to search for the optimal conditions, and then response surface methods was used for statistical analysis. We found that tryptone is an indispensable factor in cyanophycin production. Yeast extract has been upgraded to 1.37 times in optimal TB medium. Potassium phosphate plays a negative correlation with cyanophycin yield. Therefore, the optimal TB medium contains 2.4% tryptone, 3.28% yeast extract, 0.4% glycerol, 50 mM aspartic acid, 50 mM arginine, and induction with 0.2% of rhamnose under 100 rpm, 20℃ for 24 hours, which can lead to a higher yield of cyanophycin.

Keywords：cyanophycin, medium optimiztion, response surface method.

摘要…………………………………………………………………………………………………………………………………………………I
Abstract……………………………………………………………………………………………………………………………………II
目錄…………………………………………………………………………………………………………………………………………………Ⅲ
圖目錄……………………………………………………………………………………………………………………………………………Ⅳ
表目錄……………………………………………………………………………………………………………………………………………Ⅴ
附錄目錄…………………………………………………………………………………………………………………………………………Ⅵ
壹、 前言……………………………………………………………………………………………………………………………………1
貳、 文獻整理……………………………………………………………………………………………………………………………2
1. 藍藻蛋白介紹………………………………………………………………………………………………………………………2
1-1藍藻蛋白的發現及其生物功能…………………………………………………………………………………………2
1-2藍藻蛋白的結構與特性………………………………………………………………………………………………………2
1-3藍藻蛋白的運用價值……………………………………………………………………………………………………………2
2. 藍藻蛋白合成酶介紹……………………………………………………………………………………………………………3
3. Synechocystis sp. PCC6803 菌株介紹……………………………………………………………3
4. 宿主菌株介紹…………………………………………………………………………………………………………………………4
4-1. CleanColi BL21(DE3) 宿主菌株介紹………………………………………………………………4
4-2. E. coli BL21(DE3)-CodonPlus-RIL 宿主菌株介紹………………………………5
5. 反應曲面法 (response surface methods，RSM)…………………………………………5
5-1. 五水準因子設計 (five-level factorial design)………………………………5
5-2. 反應曲面法最適化步驟…………………………………………………………………………………………………6
5-3. 陡升路徑法 (Method of steepest descent)…………………………………………6
5-4. 反應曲面之適切性檢驗…………………………………………………………………………………………………7
6. ImageJ 軟體分析原理……………………………………………………………………………………………………8
参、 實驗設計與流程 ……………………………………………………………………………………………………9
肆、 實驗材料……………………………………………………………………………………………………………………………12
1. 實驗菌株與載體…………………………………………………………………………………………………………………12
1-2 表現載體……………………………………………………………………………………………………………………………12
1-3 實驗菌株……………………………………………………………………………………………………………………………12
2. 抗生素 …………………………………………………………………………………………………………………………………12
3. 標準品 …………………………………………………………………………………………………………………………………12
3-1蛋白質標準品………………………………………………………………………………………………………………………12
3-2 DNA標準品…………………………………………………………………………………………………………………………12
4. 市售套組………………………………………………………………………………………………………………………………12
5. 化學藥品………………………………………………………………………………………………………………………………12
6. 實驗設備………………………………………………………………………………………………………………………………13
伍、 實驗方法……………………………………………………………………………………………………………………………15
1. pRham-cphA 轉形至宿主 CleanColi BL21(DE3)之蛋白質表現………………15
1-1 製備小量質體 DNA…………………………………………………………………………………………………………15
1-2 DNA 濃度之定量……………………………………………………………………………………………………………15
1-3 製備 CleanColi BL21(DE3) 電穿孔勝任細胞………………………………………………15
1-4 電穿孔轉形作用 (electrotransformation)………………………………………………16
1-5 Colony PCR篩選轉形株……………………………………………………………………………………………16
1-6 DNA 洋菜膠電泳分析……………………………………………………………………………………………………17
1-7 菌種保存……………………………………………………………………………………………………………………………17
1-8 最佳 rhamnose 誘導濃度試驗………………………………………………………………………………17
1-9 蛋白質膠電泳分析…………………………………………………………………………………………………………17
2. pRham-cphA 轉形至宿主 CleanColi BL21(DE3)-CodonPlus 之蛋白質表現………………………………………………………………………………………………………………………………………………19
2-1 製備小量質體 DNA………………………………………………………………………………………………………19
2-2 DNA 濃度定量…………………………………………………………………………………………………………………19
2-3 電穿孔轉形作用 (electrotransformation)………………………………………………20
2-4 Colony PCR 篩選轉形株…………………………………………………………………………………………20
2-5 DNA 洋菜膠電泳分析……………………………………………………………………………………………………20
2-6 製備 CleanColi BL21(DE3)-CodonPlus 電穿孔勝任細胞……………………20
2-7 電穿孔轉形作用 (electrotransformation)………………………………………………20
2-8 Colony PCR 篩選轉形株…………………………………………………………………………………………20
2-9 DNA 洋菜膠電泳分析……………………………………………………………………………………………………20
2-10 菌種保存…………………………………………………………………………………………………………………………20
2-11 最佳 rhamnose 誘導濃度試驗……………………………………………………………………………21
2-12 蛋白質膠電泳分析………………………………………………………………………………………………………21
3. pET-21b-cphA 及 pET-21b-His-cphA 轉形至宿主 CleanColi BL21(DE3) 及 CleanColi BL21(DE3)-CodonPlus 之蛋白質表現……………………………………………………………………………………………………………………………………………………21
3-1 製備小量質體 DNA…………………………………………………………………………………………………………21
3-2 DNA 濃度定量…………………………………………………………………………………………………………………21
3-3 電穿孔轉形作用 (electrotranformation)…………………………………………………21
3-4 Colony PCR 篩選轉形珠…………………………………………………………………………………………21
3-5 DNA 洋菜膠電泳分析……………………………………………………………………………………………………21
3-6 菌種保存……………………………………………………………………………………………………………………………21
3-7 最佳 IPTG 誘導濃度試驗……………………………………………………………………………………………21
3-8 蛋白質膠電泳分析……………………………………………………………………………………………………………22
4. pRham-cphA 轉形至宿主 E. coli BL21(DE3)-CodonPlus-RIL 之蛋白質表現……………………………………………………………………………………………………………………………………………22
4-1 製備小量質體 DNA…………………………………………………………………………………………………………22
4-2 DNA 濃度定量…………………………………………………………………………………………………………………22
4-3 電穿孔轉形作用 (electrotranformation)…………………………………………………22
4-4 Colony PCR 篩選轉形珠…………………………………………………………………………………………22
4-5 DNA 洋菜膠電泳分析……………………………………………………………………………………………………22
4-6 菌種保存……………………………………………………………………………………………………………………………22
4-7 最佳 rhamnose 誘導濃度試驗…………………………………………………………………………………22
4-8 蛋白質膠電泳分析……………………………………………………………………………………………………………23
5. TB 培養基組成對於藍藻蛋白在 E. coli BL21(DE3)-CodonPlus-RIL 中產量之最適化探討…………………………………………………………………………………………………………………23
5-1 TB 培養基單一因子實驗測試………………………………………………………………………………………23
5-2 以反應曲面法進行 TB 培養基最適化分析………………………………………………………………23
5-3 以最適化 TB (TB-OPT) 培養基培養含有 pRham-cphA 之 E. coli BL21(DE3)-CodonPlus-RIL 生長與誘導條件分析…………………………………………………23
5-3-1 最佳 rhamnose 誘導濃度試驗……………………………………………………………………………23
5-3-2 蛋白質膠電泳分析………………………………………………………………………………………………………24
5-3-3 最佳 amino acid 濃度測試………………………………………………………………………………24
5-3-4 蛋白質膠電泳分析………………………………………………………………………………………………………24
5-4 藍藻蛋白之純化………………………………………………………………………………………………………………24
5-4-1 藍藻蛋白之純化前處理……………………………………………………………………………………………24
5-4-2水溶性 (water-soluble type) 藍藻蛋白純化法……………………………………25
5-4-3 非水溶性 (water-insoluble type) 藍藻蛋白純化法………………………25
5-4-4 細胞乾重 (dry cell weight)………………………………………………………………………25
5-4-5 分析藍藻蛋白之分子量大小……………………………………………………………………………………25
陸、結果與討論…………………………………………………………………………………………………………………………26
柒、結論………………………………………………………………………………………………………………………………………32
捌、參考文獻………………………………………………………………………………………………………………………………33

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