臺灣博碩士論文加值系統

本研究在於探討創傷弧菌藍色螢光蛋白基因 (bfp) 與經由DNA變異法所得突變基因 (bfp-d7) 在革蘭氏陰性菌Escherichia coli XL1-Blue與Vibrio vulnificus CKM-1巨大原生質體之螢光表現，並利用巨大原生質體評估重組基因啟動子強度之方法。

實驗中定義兩種螢光值，分別為比螢光值與總螢光值，比螢光值為去單位化Image-Pro Plus所分析出螢光色階之數據，而總螢光值為比螢光值乘上光學密度(Optical Density 600)，可正規化比螢光值，以利起動子效率的估算。啟動子效率偵測方面，共有三種變數，每種變數有兩個參數，分別為菌株 (E.coli XL1-Blue、V.vulnificus CKM-1)、螢光蛋白 (BFP、BFP-D7) 與溫度 (30°C、25°C)，搭配起來有8種變化，並根據總螢光值對時間之曲線的最高點到最低點範圍，以線性趨勢線逼近，得趨勢線斜率，代表啟動子效率。比較以上各種變化之啟動子效率，得到一偵測啟動子強度之最佳系統為利用菌株E.coli XL1B在BFP-D7螢光蛋白表現下，於30°C偵測啟動子強度。

The expression of blue fluorescent protein gene (bfp) and mutated gene (bfp-D7) from bfp by random mutagenesis isolated from Vibrio vulnificus CKM-1 in the giant protoplasts of Escherichia coli XL1B and V. vulnificus CKM-1 were investigated, respectively. In addition, we built a system for quantifying the fluorescent intensity of BFP and screened a high efficiency promoter.

We defined two kinds of fluorescence density, one was specific blue density that was de-unit data from Image-Pro Plus’ color depth ; another’s overall blue density that was from specific blue density multiplied optical density. The overall blue density was able to represent specific blue density normalization and evaluated transcription efficiency in promoter. About promoter efficiency evaluated, we had three kinds of variables including two parameters with eight variations, one was bateria including E.coli XL1-Blue and V. vulnificus CKM-1, one was fluorescence protein including BFP and BFP-D7, another was temperature including 30°C and 25°C. According the diagram of overall blue density varied with time, we found the slope of regression line fitting the curve which range was from the highest fluorescence value to the lowest value. The slope represented promoter efficiency. The results showed that the most adaptable system to detect promoter intensity was E.coli XL1-Blue_pUD7 in 30°C.

目錄

中文摘要　I
英文摘要　II
誌謝　III
目錄　IV
表目錄　VII
圖目錄　VIII
符號對照表　XI

第一章　緒論　1
1-1　前言　1
1-2　細菌細胞外部構造簡介　2
1-2-1　細菌細胞外部構造　2
1-2-2　革蘭氏陰性菌外部構造　3
1-3　原生質體簡介　10
1-4　革蘭氏陰性菌原生質體形成方法　10
1-5　生物冷光蛋白　11
1-5-1　水母 (Aequorea Victoria) 綠色螢光蛋白　13
1-5-2　創傷弧菌 (Vibrio vulnificus) 藍色螢光蛋白　14
1-6　報導基因 (reporter gene)　16
1-7　生物螢光影像　18
1-8　研究動機與目的　18
第二章　實驗材料與方法　20
2-1　實驗材料　20
2-1-1　藥品　20
2-1-2　實驗儀器　21
2-1-3　菌株　22
2-1-4　載體　22
2-1-5　培養基、緩衝液與Stock Solution　23
2-2　實驗方法　26
2-2-1　菌種保存與活化　26
2-2-2　原生質體之製備與培養　26
2-2-3　大腸桿菌之轉型作用　27
2-2-4　製備少量質體DNA　28
2-2-5　原生質體之螢光表現定量與圖像分析　29
2-2-6　細胞計數　30
第三章　結果與討論　31
3-1　原生質體的特性　31
3-1-1　原生質體大小之擷取　31
3-1-2　原生質體的構造　32
3-1-3　V.vulnificus CKM-1菌體大小與生長曲線　33
3-1-4　E.coli XL1B菌體大小與生長曲線　39
3-2　質體構築　44
3-2-1　pUD7載體之構築　44
3-3　螢光取像系統的建立　46
3-3-1　亮區選取　46
3-3-2　螢光亮度值的建立　47
3-3-3　環境螢光背景值的建立　48
3-3-4　原生質體螢光背景值的建立　50
3-3-5　一分鐘螢光衰退半衰期的確立　50
3-3-6　估算啟動子 (Promoter) 效率方法之確立　51
3-4　V.vulnificus CKM-1菌株之螢光表現　53
3-4-1　CKM-1_pUVF1原生質體螢光亮度之變化　53
3-4-2　CKM-1_pUD7原生質體螢光亮度之變化　53
3-5　E.coli XL1B菌株之螢光表現　66
3-5-1　XL1B_pUVF1原生質體螢光亮度之變化　66
3-5-2　XL1B_pUD7原生質體螢光亮度之變化　66
3-6　啟動子效率之比較　80
3-6-1　30°C與25°C啟動子效率之比較　80
3-6-2　BFP-D7與BFP啟動子效率之比較　81
3-6-3　CKM-1與XL1B啟動子效率之比較　81
第四章　結論與未來展望　85
參考文獻　87
自述　91

表目錄

表1-2-1　革蘭氏陽性菌與革蘭氏陰性菌的比較　5
表1-5-1　改質之綠色螢光蛋白　16
表1-6-1　Reporter Gene　17
表2-1-1　菌株　22
表2-1-2　載體　22
表2-1-3 (a)　LB培養基組成　23
表2-1-3 (b)　GP700培養基組成　23
表2-1-3 (c)　SP buffer組成　24
表2-1-3 (d)　Solution stoke　24
表2-2-1 (a)　大腸桿菌轉型用試劑　27
表2-2-1 (b)　製備少量質體DNA用試劑　28
表3-3-1　螢光亮度值計算方法　47
表3-6-1　CKM-1總螢光趨勢線之數據　83
表3-6-2　CKM-1 promoter效率之比較順序　83
表3-6-3　XL1B總螢光趨勢線之數據　84
表3-6-4　XL1B promoter效率之比較順序　84

圖目錄

圖1-2-1　膜構造　6
圖1-2-2　革蘭氏陰性菌與革蘭氏陽性菌細胞壁構造之區別　7
圖1-2-3　細菌細胞外部構造　8
圖1-2-4　Peptidoglycan之結構　9
圖1-4-1　原生質體形成步驟圖　11
圖1-5-1　激發電子能階圖　15
圖1-5-2　冷光細菌細胞內藉由luciferase在氧氣存在下之發光機制　15
圖1-5-3　綠色螢光蛋白立體結構與激發、放射光譜圖　15
圖1-5-4　改質之綠色螢光蛋白激發與放射光譜圖　16
圖2-1-1　pUC19 基因圖譜　25
圖2-2-1　血球計數器counting chamber，放大100倍　30
圖3-1-1　細胞影像之擷取　31
圖3-1-2　CKM-1 puD7原生質體形態　32
圖3-1-3　CKM-1正常菌體生長曲線　35
圖3-1-4　CKM-1原生質體生長曲線　36
圖3-1-5　CKM-1_pUVF1菌體濃度與大小　37
圖3-1-6　CKM-1_pUD7菌體濃度與大小　38
圖3-1-7　XL1B正常菌體生長曲線　40
圖3-1-8　XL1B原生質體生長曲線　41
圖3-1-9　XL1B_pUVF1菌體濃度與大小　42
圖3-1-10　XL1B_pUD7菌體濃度與大小　43
圖3-2-1　pUD7質體圖譜　44
圖3-3-1　XL1B_pUD7-10hr (30) 1分鐘色階分佈與背景螢光亮度圖　49
圖3-3-2　XL1B host 原生質體螢光亮度值與半徑關係圖　52
圖3-4-1　CKM-1_pUVF1 (30) 2~16小時原生質體螢光照片、放大400倍　55
圖3-4-2　CKM-1_pUVF1 (25) 2~16小時原生質體螢光照片、放大400倍　56
圖3-4-3　CKM-1_pUVF1原生質體螢光亮度圖　57
圖3-4-4　CKM-1_pUD7_2hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　58
圖3-4-5　CKM-1_pUD7_4hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　59
圖3-4-6　CKM-1_pUD7_6hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　60
圖3-4-7　CKM-1_pUD7_8hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　61
圖3-4-8　CKM-1_pUD7_10hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　62
圖3-4-9　CKM-1_pUD7_12hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　63
圖3-4-10　CKM-1_pUD7_16hr原生質體放大400倍螢光照片與1分鐘衰退趨勢圖　64
圖3-4-11　CKM-1 pUD7原生質體螢光亮度圖　65
圖3-5-1　XL1B_pUVF1 (30) 2~16小時原生質體螢光照片與37°C 正常菌體螢光照片、放大400倍　68
圖3-5-2　XL1B_pUVF1 (25) 2~16小時原生質體螢光照片、放大400倍　69
圖3-5-3　XL1B_pUVF1原生質體螢光亮度圖　70
圖3-5-4　XL1B_pUD7-2hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　71
圖3-5-5　XL1B_pUD7-4hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　72
圖3-5-6　XL1B_pUD7-6hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　73
圖3-5-7　XL1B_pUD7-8hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　74
圖3-5-8　XL1B_pUD7-10hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　75
圖3-5-9　XL1B_pUD7-12hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　76
圖3-5-10　XL1B_pUD7-16hr原生質體與正常菌體放大400倍螢光照片與1分鐘螢光衰退趨勢圖　77
圖3-5-11　XL1B_pUD7螢光灰度圖　78
圖3-5-12　添加NADPH對CKM-1 pUD7 (25)原生質體螢光之影響　79
圖3-6-1　CKM-1 啟動子效率圖　83
圖3-6-2　XL1B 啟動子效率圖　84

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