臺灣博碩士論文加值系統

本論文利用基因重組技術建立以whole-cell為主之偵測系統，此生物偵測器之重組質體(pVLCD1)主要藉由pI258質體中之cadC基因調控綠色螢光蛋白之表達。本研究之E. coli生物偵測器對於金屬鎘、鉛、銻及鋅產生螢光反應，其最低偵測濃度分別為100nM, 100nM, 100nM及1μM，螢光強度與各金屬之濃度成正相關。其它金屬方面如:砷、鈷、銅、鐵、汞、錳、鎳及錫對於本研究之生物偵測器並無顯著誘導螢光之產生。

A whole cell sensing system had been designed and developed using genetically engineered bacteria. The sensor plasmid, pVLCD1, was constructed by inserting the regulation unit of the cadC determinant from plasmid pI258 to control the expression of GFP. The Escherichia coli biosensor could detect the presence of cadmium (Cd2+), lead (Pb2+), antimony (Sb2+) and zinc (Zn2+), and the lowest responding concentrations for these metals was,100nM, 100nM, 100nM, and 1μM ,respectively. The strength of the fluorescence intensity was related to the concentration of metal ions. The biosensor showed no fluorescence to other metal ions (As3+、Co2+、Cu2+、Fe2+、Hg2+、Mn2+、Ni2+、Sn2+).

目 錄
誌謝………………………………………………………………………….. .I
英文摘要…………………………………………………………………….. II
中文摘要……………………………………………………………………. III
目錄…………………………………………………………………………..IV
表目錄………………………………………………………………………VIII
圖目錄………………………………………………………………………...IX
縮寫表………...……………………………………………….…………….XII
第一章 研究緣起與目的
1.1研究緣起………………………………………………………………1
1.2研究目的………………………………………………………………1
第二章 文獻回顧
2.1前言……………………………………………………………………3
2.2生物偵測器……………………………………………………………4
2.3綠色螢光蛋白…………………………………………………………5
2.4 Cad operon……………………………………………………………8
2.5不同重金屬Biosensor之探討………………………………………10
2.5.1鎘(Cd)………………………………………………………….10
2.5.2汞(Hg)………………………………………………………….11
2.5.3砷(As)…………………………………………………………. 12
2.5.4其它…………………………………………………………… 14
第三章 實驗材料與方法
3.1實驗流程……………………………………………………………..16
3.2菌種與質體…………………………………………………………..18
3.3實驗藥品……………………………………………………………..18
3.3.1培養基……………………………………………………….. 18
3.3.2核酸電泳…………………………………………………….. 18
3.3.3Miniprep…….………………………………………………. 19
3.3.4PCR…….……………………………………………………. 19
3.3.5Compontent cell and transformation…...…………………..19
3.3.6Double digest…………………………………………………19
3.3.7Ligation…….…………………………………………………19
3.3.8Gel extraction and Purification……………………………..20
3.3.9重金屬測試……………………………………………….. …20
3.4實驗器材……………………………………………………………..20
3.4.1培養基部份………………………………………………….. 20
3.4.2核酸電泳部份……………………………………………….. 20
3.4.3Miniprep部份……………………………………………….. 20
3.4.4PCR部份…………………………………………………….. 20
3.4.5Compontent cell and transformation…...…………………. 20
3.4.6 Gel extraction and Purification…………………………… 20
3.4.7生長曲線……………………………………………………...21
3.4.8其它…………………………………………………………...21
3.5實驗步驟……………………………………………………………..21
3.5.1Miniprep……………………………………………………...21
3.5.2PCR…….……………………………………………………..22
3.5.3 Doubledigest…………………………………………………24
3.5.4Ligation…….…………………………………………………25
3.5.5Compontent cell and transformation…..…………………...25
3.5.6各金屬之螢光強度測試……………………………………..26
3.5.7各金屬之毒性效應…………………………………………..27
3.5.8劑量反應及誘導表達實驗…………………………………. 27
3.5.9即時發光實驗………………………………………………. 28
3.6數據之計算………………………………………………………… 29
3.6.1各金屬之螢光強度測試……………………………………..29
3.6.2 各濃度之p-value之計算………………………………….. 29
3.6.3 誘導速率常數之計算……………………………………….29
第四章 實驗結果………………………………………………………….. 31
4.1重組質體pVLCD-1…………………………………………….......31
4.1.1萃取pI258質體……………………………………………. 31
4.1.2萃取pBROBE-NT’質體…………………………………… 31
4.1.3PCR………………………………………………………….. 32
4.1.4Double digestion pBROBE-NT’質體…………………….....33
4.1.5確認重組質體pVLCD-1……………………………………33
4.2各種重金屬之螢光強度測試……………………………………….34
4.3各金屬之毒性測試………………………………………………….36
4.3.1鎘(Cd2+)………………………………………………………36
4.3.2鉛(Pb2+)……………………………………………………….37
4.3.3銻(Sb2+)……………………………………………………….38
4.3.4鋅(Zn2+)………………………………………………………39
4.4 鎘、鉛、銻及鋅之劑量與速率反應實驗結果…………………….39
4.4.1.1劑量反應(鎘) ………………………………………………40
4.4.1.2速率反應(鎘) ………………………………………………44
4.4.2.1劑量反應(鉛) ………………………………………………45
4.4.2.2速率反應(鉛) ………………………………………………49
4.4.3.1劑量反應(銻) ………………………………………………50
4.4.3.2速率反應(銻) ………………………………………………54
4.4.4.1劑量反應(鋅) ………………………………………………55
4.4.4.2速率反應(鋅) ………………………………………………59
4.5鎘、鉛、銻與鋅之誘導比較……………………………………….59
4.6金屬鎘與銻之動力學實驗結果…………………………………….60
4.7即時發光偵測與生長時期發光影響之實驗結果………………….61
第五章 討論與建議………………………………………………………….64
5.1討論………………………………………………………………….64
5.2建議………………………………………………………………….69
第六章 參考文獻…………………………………………………………….70

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