利用基因改造工程優化大腸桿菌BL21對間二甲苯之偵測能力

Enhanced detection of m-xylene by engineering Escherichia coli BL21

顏家禾(Yen Chia-Ho)

指導教授 ： 葉怡均

國立臺灣師範大學/理學院/化學系/碩士(2016年)

https://doi.org/10.6345/NTNU202204365

摘要

本研究利用基因改造工程，擷取戀臭假單胞菌 (Pseudomonas putida)TOL質體中的xyl調控組，並嵌入人工質體中。當大腸桿菌BL21感測到環境中的間二甲苯 (m-xylene)，XylR蛋白會與間二甲苯結合形成複合物，此複合物會觸發下游啟動子，開始轉錄形成報導蛋白。本研究借助兩種蛋白作為訊號輸出：一是紅色螢光蛋白 (RFP)；二是顯色明顯的紫茉莉4,5多巴雙加氧酶 (MJDOD)，能將左旋多巴催化形成甜菜色素。透過對TOL網絡 (TOL network)調節機轉的了解，我們以一系列人工質體進行實驗，除了置換啟動子，利用不同報導蛋白進行檢測也是研究方向之一。經過實驗比較與佐證，我們發現其中一個菌株針對間二甲苯具有不錯的偵測結果，訊號線性範圍從0至1600μM，也具有一定的專一性。為了增加在野外進行環境分析的實用性，我們以瓊脂糖 (agarose)包覆菌液形成菌珠，可省略實驗前細菌培育步驟。綜合上述特點，本研究為有機溶劑污染檢測方式，增添一個可能性。

關鍵字

TOL網絡 ； XylR蛋白 ； 微生物感測器 ； 戀臭假單胞菌 ； 大腸桿菌 ； 環境分析方法

並列摘要

This thesis focuses on utilizing xyl gene cluster to engineer an artificial plasmid. When engineered Escherichia coli BL21 strain senses m-xylene in environment, XylR protein binds to m-xylene and forms a complex. The complex triggers promoter of reporter protein, then reporter protein will be transcribed. We employed two signal outputs, one is red fluorescence protein (RFP), the other is Mirabilis jalapa DOPA 4,5-dioxygenase (MJDOD). RFP is a fast-folding protein and betaxanthin has brilliant color, which is synthesized from L-3,4-dihydroxyphenylalanine (L-DOPA) by MJDOD. We design various constructs in order to enhance detection performance based on the regulation mechanism of TOL network. The best performance strain was screened and examined, which was specific to m-xylene and had detection linear range: 0~1600μM. For portable application, our biosensor was coated by agarose to form bacterial beads. Bacterial beads can detect m-xylene without complex incubation procedures, which saves a lot of time. With these advantages, XylR-based biosensor can be an alternative detection option for organic solvents

並列關鍵字

TOL network ； XylR protein ； biosensor ； Pseudomonas putida ； Escherichia coli ； environmental analysis

參考文獻

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