以Lpp-OmpA工法建構新穎性基因工程菌強化鎘生物復育能力

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姓名

何欣澄(Hsin-Cheng Ho) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

以Lpp-OmpA工法建構新穎性基因工程菌強化鎘生物復育能力
(Constructing novel engineered bacteria based on Lpp-OmpA system to enhance the cadmium bioremediation)

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★ 研究雙特松對HepG2細胞之DNA修復的影響	★ 金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試

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摘要(中)

隨著工業的發展，工廠迅速地在各處被建立，疏於管理的情況下，造成許多重金屬污染隨之發生。重金屬具有毒性且無法被分解，能夠累積於人體內，導致疾病的發生。本研究試圖以膜上表現系統將金屬硫蛋白表現於大腸桿菌外膜上，藉此提升菌體對重金屬的生物復育能力。首先，利用基因編輯技術，將金屬硫蛋白與膜上表現系統Lpp-OmpA融合，藉此表現金屬硫蛋白於細胞表面，以改善金屬離子被阻擋在細胞膜外，無法被菌體吸附的阻礙。與此同時，本研究評估基因轉殖菌在大量表現蛋白質時最適合的條件。接著利用西方墨點法及綠色螢光蛋白，確認目標蛋白的表現與摺疊。轉殖菌株建構完成後，針對E. coli C43(DE3) pLysS以及攜帶pLO及pLM5載體的該菌株進行對重金屬鎘的敏感性測試，在達80 mg/L鎘離子濃度時，表現pLM5使其相較其他菌株具有較高抗性。在鎘吸附測試中，基因轉殖菌表現LO-MTT5於膜上之菌體吸附容量為1.47 mg/g，相較胞內表現MTT5之吸附容量0.2585 mg/g提升5.6倍的吸附能力，其改善重金屬被阻隔在外而對吸附造成的障礙。在不同濃度鎘離子吸附測試中，發現隨著環境中鎘離子濃度由2.5 mg/L提升至80 mg/L，吸附量會隨之上升，然而移除率卻由54.8%降至24.6%。在Langmuir及Freundlich等溫模式中，前者qm為3.1308 mg/g、KL為0.0267、RL為0.3192，後者KF為0.0677、n為1.0138，皆顯示為有效吸附，並由R2值推測該吸附模式較符合多層吸附。在FTIR放射光譜，N-H、C=O、P=O鍵結波長產生位移，代表這些鍵結參與鎘離子的吸附。在mRNA基因表現分析中，表現pLM5時dnaK、clpB基因不受鎘離子存在影響，推得LO-MTT5對菌體形成保護，使其蛋白質能正常摺疊。另外，czcB基因受鎘離子影響而表現量上升，顯示鎘離子存在會使菌體表現大量鎘外排幫浦，可能為降低胞內累積的原因之一。最後在污水的離地測試中，顯示基因轉殖菌株吸附能力不受其他金屬離子影響，並且在48小時達到20.5 mg/g的鎘離子吸附量，具有應用於現地的潛力，未來可以針對其回收作更深入之研究。

摘要(英)

Accroding to the industrial delopment, factories were rapidly built. Due to the neglect of management, heavy metal pollution occurred. Heavy metals, such as Cadmium (Cd), is toxic and non-degratable, which accumulate inside the human body resulting in disease. In our study, the surface display system Lpp-OmpA was fused to the metal-binding protein metallothionein to enhance bioremediation. After construction, the suitable condition for protein overexpression was evaluated. Western blot and green fluorescent protein were used to confirm the expression and folding of the target protein. In the cadmium sensitivity test, E. coli with pLM5 has higher resistance than other strains in 80 mg/L Cd2+. In the cadmium adsorption test, the adsorption capacity of the E. coli with pLM5 was 1.47 mg/g. Compared with the adsorption capacity of E. coli with pET-MTT5 (0.2585 mg/g), the adsorption capacity of E. coli with pLM5 has shown a 5.6-fold impovement. The result showed that outer membrane display stradegy was able to overcome the obstacles in cadmium adsorption. For increased initial Cd2+ concentrations of 2.5 - 80 mg/L, the adsorption capacity would increase, but the removal rate would drop from 54.8% to 24.6%. Otherwise, biosorption data of E. coli expressing pLM5 preferred to be simulated with Freundlich model. In Langmuir and Freundlich isotherm models, the correlation parameters for each model were shown. In the FTIR emission spectrum, the wavelengths of the N-H, C=O, and P=O bonding have shifted, indicating that these bonds are involved in the adsorption of Cd2+. In the mRNA gene expression analysis, the dnaK and clpB genes indicated the expression of LO-MTT5 may inhibit protein misfolding. In addition, the expression of czcB gene indicate that the presence of Cd2+ would induce the expression of cadmium efflux pump. Finally in the real wastewater test, the Cd2+ adsorption capacity of E. coli with pLM5 reached 20.5 mg/g within 48 hours. In conclusion, the outer membrane expression of metallothionein was shown as a potential techenique for recover industrial pollution.

關鍵字(中)

★ 鎘
★ 生物復育
★ 基因轉殖
★ 金屬硫蛋白
★ 膜上表現

關鍵字(英)

★ Cadmium
★ Bioremediation
★ Transgene
★ Metallothionein
★ Surface display

論文目次

致謝 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 vii
表目錄 ix
公式目錄 ix
第壹章緒論 (Introduction) 1
1.1 研究背景 1
1.2 鎘金屬的特性與危害 2
1.3 生物復育 (Bioremediation) 2
1.3.1 微生物策略 (Microbial remediation) 3
1.3.2 生物吸附與生物累積 3
1.4 基因轉殖 4
1.4.1 表現宿主：大腸桿菌 (Escherichia coli) 5
1.4.2 膜上表現系統：Lpp-OmpA 6
1.4.3 金屬硫蛋白：四膜蟲之金屬硫蛋白 MTT5 7
1.5 基因轉殖菌之應用與價值 8
第貳章實驗目的與實驗架構 9
第參章實驗材料與方法 (Meterials and Methods) 11
3.1 實驗材料 11
3.1.1 使用儀器與廠牌 11
3.1.2 常用藥品與試劑 13
3.2 實驗方法 17
3.2.1 菌株保存與繼代培養 (E. coli) 17
3.2.2 細菌質體萃取 18
3.2.3 聚合酶連鎖反應(Polymerase chain reaction ,PCR) 18
3.2.4 TSS 法之大腸桿菌勝任細胞製備及轉型 19
3.2.5 限制酵素處理 21
3.2.6 DNA 膠體純化 22
3.2.7 DNA 接合反應 22
3.2.8 蛋白質誘導 23
3.2.9 蛋白質萃取 24
3.2.10 膜蛋白分離萃取 24
3.2.11 蛋白質濃度檢定(Bradford 法) 25
3.2.12 蛋白質濃度檢定(RC-DC 法) 26
3.2.13 SDS-PAGE 蛋白質電泳 26
3.2.14 西方免疫墨點法 (Western blot) 28
3.2.15 細菌生長曲線測定 29
3.2.16 重金屬吸附試驗 30
3.2.17 細菌 RNA 萃取 31
3.2.18 反轉錄作用 (Reverse transcription) 32
3.2.19 即時定量連鎖聚合酶反應 (Real-time quantitative polymerase chain reaction, Q-PCR) 33
第肆章實驗結果 (Result) 34
4.1 建構基因轉殖菌 C43(DE3) pLysS/pLM5 34
4.1.1 建構膜上表現載體 pLO 及 pLM5 34
4.1.2 融合蛋白 LO-MTT5 誘導表現測試 35
4.1.3 藉由螢光蛋白及膜蛋白分離萃取以驗證蛋白質成功表現 35
4.2 研究基因轉殖菌 C43(DE3) pLysS/pLM5 對鎘金屬的吸附 37
4.2.1 檢驗基因轉殖菌 C43(DE3) pLysS/pLM5 之重金屬敏感度 37
4.2.2 比較金屬硫蛋白 MTT5 表現於不同位置對鎘離子吸附能力的影響 37
4.2.3 鎘離子暴露濃度對基因轉殖菌 C43(DE3) pLysS/pLM5 鎘離子吸附能力之影響 38
4.2.4 等溫吸附模式 38
4.3 探討表現融合蛋白 LO-MTT5 在 E. coli 外膜上相關的生物機制 40
4.3.1 以 FTIR 光譜法分析鎘離子與蛋白質的鍵結關係 40
4.3.2 以 qPCR 分析 C43(DE3)/pLM5 抵禦鎘之相關基因表達 40
4.4 工業污染水之離地測試 42
第伍章討論 (Discussion) 43
5.1 融合蛋白 Lpp-OmpA-MTT5 之設計及表現 43
5.2 整合螢光蛋白 sfGFP 以檢視蛋白質表現 44
5.3 膜上表現金屬硫蛋白 MTT5 加強對鎘的生物吸附 45
5.4 基因轉殖菌 C43(DE3) pLysS/pLM5 之生物復育機制探討 48
5.5 應用基因轉殖菌進行污染水離地測試 50
第陸章結論 (Conclusion) 51
參考文獻 (Reference) 52
圖表 60
附加資料 (Supplementary file) 79

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指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2021-7-27

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