臺灣博碩士論文加值系統

測量水中微生物含量有很多方法，其中較快速且靈敏的為冷光偵測法。藉由冷光偵測法可將生物體產生的腺苷三磷酸(ATP)轉換成光，再利用冷光儀測量，即可換算得知水中細菌含量。目前商品化產品可以偵測的微生物極限濃度約在104 CFU/ml ，為了提高偵測靈敏度，本計畫利用大腸桿菌的腺苷二磷酸-葡萄糖焦磷酸酶(ADP-glucose pyrophosphorylase) ，在ADPglucose存在下，與冷光酵素的產物焦磷酸反應，生成ATP與葡萄糖一磷酸(glucose-1-phosphate)，形成一個ATP再循環系統，以提高冷光的訊號。此ATP再循環系統的另一優點是比市面上用在焦磷酸測序(pyrosequencing)技術上轉換PPi為ATP的腺苷三磷酸硫酸化酶/五硫磷酸腺苷系統(ATP sulfurylase/adenosine 5´ phosphosulfate system)有較低的背景值。這套ATP再循環系統不管在液態培養基中的大腸桿菌，綠膿桿菌及仙人掌桿菌或是這些菌種形成的生物膜偵測上都有所提昇，能將微生物偵測極限降低至103 CFU/ml。為了更進一步要再提高微生物偵測的靈敏度，我們提出了雙途徑ATP增幅系統 (Bi-directional ATP amplification)的概念，本方法除了利用ATP sulfurylase/adenosine 5´ phosphosulfate轉換PPi為ATP外，同時利用腺苷酸激酶（adenylate kinase）將尿苷三磷酸(UTP)做為能量提供者把發光酵素的另一個產物腺苷單磷酸(AMP)轉為腺苷雙磷酸(ADP)，再利用醋酸激酶(acetate kinase)在乙醯磷酸(acetyl phosphate)存在下將ADP轉成ATP，藉由兩邊同時再生ATP的形式，穩定增加ATP的量以大幅提高冷光訊號的效果，克服ATP sulfurylase/adenosine 5´ phosphosulfate system產生的背景值，並達到102 CFU/ml 的微生物偵測極限。而ATP再循環系統除了可以提昇水中微生物偵測的效果外，未來也可運用於分子診斷相關的檢測中，迅速放大反應產物焦磷酸以得到冷光訊號。

The manufacturing processes of many electronic and medicinal products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilm. Several bacteria detection methods have been employed for this purpose including ATP luminescence assay, polymerase chain reactions, fluorescent in situ hybridization, and a β-glucuronidase-based assay. Among them, the ATP luminescence assay is a rapid, sensitive and easy to perform method. The first aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate (PPi), a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PPi in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5´ phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus either in broth or biofilm, and a detection limitation of 103 CFU/ml can be achieved. The second aim of this study is to increase the detection sensitivity from 104 CFU/ml to 102 CFU/ml of bacteria. One possible approach is to regenerate ATP simultaneously from both AMP and PPi, two major products of ATP luminescence assay, in a one-tube reaction. Regenerating AMP into ATP involves two consecutive enzyme reactions: adenylate kinase converting AMP and UTP into ADP and UDP; followed by acetate kinase catalyzing acetyl phosphate and ADP into ATP and acetate. Unfortunately, the AGPPase-based assay resulted in a high luminescence background when coupled with adenylate kinase. Therefore, ATP sulfurylase/APS system was used in the AMP-PPi Bi-directional ATP amplification (BDAA) system to replace AGPPase-based assay. These findings indicate that the AGPPase-based and the AMP-PPi bi-pathway ATP regeneration systems could increase the detection limitation to 102 CFU which will find many practical applications such as detection of bacterial biofilm in water pipelines.

目錄

Abstract i
中文摘要 iii
誌謝 v
縮寫 vi
目錄 viii

前言 1

實驗材料與方法 4
菌株、質體及菌株培養條件 4
染色體DNA製備 4
質體DNA製備 5
轉型作用(熱休克法) 5
IPTG誘導基因表現 6
His tag 親和性管柱純化蛋白質 6
十二酯硫酸鈉-聚丙烯醯氨膠體電泳 (SDS-PAGE) 7
蛋白質之透析 7
蛋白質之活性測試 8
UDPglc PPase (UGPPase)參與的ATP 循環系統 8
ADPglc PPase (AGPPase)參與的ATP 循環系統 9
1. 一般ATP冷光分析及ADPglc PPase參與的冷光分析 9
2. 利用冷光分析偵測液態培養基中的細菌及其產生的
生物膜 9
BDAA (Bi-directional ATP amplification)雙循環系統 10
1. BDAA雙循環系統及其餘單循環系統參與的冷光分析
10
2. 利用冷光分析偵測液態培養基中的細菌 11
高壓液相層析 (High Pressure Liquid Chromatography) 11

實驗結果 13
實驗流程 13
將ADPglc PPase (AGPPase)基因轉殖到E. coli BL21菌株內 13
ADPglc PPase蛋白質從E. coli BL21菌株內純化的結果 14
UGPPase循環系統對於ATP冷光分析的效果 14
AGPPase循環系統對於ATP冷光分析的效果 15
比較AGPPase和ATP sulfurylase參與的循環系統對於冷光
分析的效果 16
AGPPase的活性測試及最佳化 16
AGPPase/ADPglc再循環系統在偵測不同濃度的PPi所呈現的
線性關係 17
AGPPase/ADPglc再循環系統在偵測不同濃度的ATP所呈現的
線性關係 17
AGPPase/ADPglc再循環系統對於細菌及生物膜的偵測能力 18
BDAA雙途徑循環系統和ATP-Sulfurylase/APS及Acetate
kinase/ Acetyl phosphate 單循環系統在冷光分上的差異 19
偵測雙途徑ATP増幅系統中冷光背景值的來源 20
BDAA雙途徑循環系統中反應濃度的最佳化 21
BDAA雙途徑循環系統偵測不同濃度ATP的線性關係 21
BDAA雙途徑循環系統對於不同菌種的冷光分析 22
應用AGPPase循環系統偵測PCR產物及反應濃度的最佳化 23

討論 25

參考文獻 30

表格 35

圖示 37

附錄 69

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