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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題名:	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
作者:	何欣澄;Ho, Hsin-Cheng
貢獻者:	生命科學系
關鍵詞:	鎘;生物復育;基因轉殖;金屬硫蛋白;膜上表現;Cadmium;Bioremediation;Transgene;Metallothionein;Surface display
日期:	2021-07-27
上傳時間:	2021-12-07 11:23:19 (UTC+8)
出版者:	國立中央大學
摘要:	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。;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.
顯示於類別:	[生命科學研究所 ] 博碩士論文

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