假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討

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呂哲瑋(Che-Wei Lu) 查詢紙本館藏

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假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討
(Development of Recombinant Pseudomonas sp. A46 for heavy metal bioremediation and its potential of bioaccumulation and biosorption)

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摘要(中)

生物累積及生物吸附是微生物作為抵抗外界重金屬的兩個重要的機制，可以將外界的重金屬與環境做分離、去毒性並固定在細菌體內，使得細菌進而成為極有潛力的生物整治工具。本研究嘗試在Pseudomonas sp. A46菌株中表現金屬硫蛋白，試圖提升其生物累積之能力。為了增加蛋白質表現之穩定以及避免使用抗生素，使得此菌株能夠應用在野外，本研究開發能於Pseudomonas sp. A46使用之反向篩選系統，在剔除upp基因後，將能表現綠螢光蛋白與金屬硫蛋白融合蛋白之基因片匣整合至染色體中，對染色體做PCR分析確認片匣成功植入染色體中，接著利用反轉錄PCR分析以及西方墨點法證實融合蛋白成功表現，並且連續繼代40次確定基因之穩定性。完成菌株建立後，針對A46、M01及pJBME測定各菌株對於重金屬銅和鎘之最小抑制濃度，銅皆為250ppm，鎘皆為20ppm，在敏感度測試亦得到一樣的結果，菌株對於重金屬之生長抗性沒有因基因改造而有所增加。在銅及鎘之生物累積試驗中，A46、M01及pJBME並沒有明顯差異，顯示金屬硫蛋白可能不能增加生物累積之能力。在EDTA淋洗試驗中，發現大部分的銅累積於胞內，推得此菌對於銅主要行生物累積，但是總體移除率只有2%，推得外送幫浦蛋白及生物膜構造有效阻隔銅的進入。然而，在相同實驗中，大多數之鎘卻吸附於胞外，並且總體移除率達30%，顯示此菌對於鎘主要行生物吸附，生物膜的產生有助於吸附重金屬鎘並且保護菌株。雖然金屬硫蛋白無法提升此株菌之生物累積能力，但本株菌對於重金屬鎘有著強大之生物吸附能力，未來可以針對其機制做更深入之研究。

摘要(英)

Bioaccumulation and biosorption are two important mechanisms for some microorganisms to resist heavy metals, which makes bacteria a potential bioremediation tool. In an attempt to enhance the bioaccumulation capacity of Pseudomonas sp. A46 strain, we cloned the gene of metallothionein into Pseudomonas sp. A46. We developed a genetic bacteria with counter selection system that can be used in Pseudomonas sp. A46. After removing the upp gene, the gene fragment that expresses the fusion protein. Thereafter, the minimum inhibitory concentrations (MIC) for the heavy metal copper and cadmium were determined for A46, M01(MT1 expressed by chromosome) and pJBME (expressed MT1 by plasmid). The MIC of copper was 250 ppm and the cadmium was 20 ppm. With these MIC values, the resistance of the genetic strain to heavy metals was not increased. In the copper and cadmium bioaccumulation assay, there was no significant difference among A46, M01, and pJBME, suggesting that metallothionein may not increase the bioaccumulation capacity. In the EDTA leaching test for the biosorption of heavy metals, most of the copper was found to accumulate in the interior of the cells. The bioaccumulation of this bacterium was a key player. However, the overall removal rate was only 2%. n the other hand, most of the cadmium was adsorbed extracellularly, and the overall removal rate was 35%, indicating that the bacterium bioremediate cadmium via biosorption, and the biofilm production was helpful in adsorbing cadmium and protecting the strain. Although metallothionein cannot increase the bioaccumulation capacity of this strain, this strain of bacteria has a strong biosorption capacity for cadmium.

關鍵字(中)

★ 銅
★ 鎘
★ 微生物整治
★ 金屬硫蛋白
★ 生物累積
★ 生物吸附

關鍵字(英)

★ Copper
★ Cadmium
★ Metallothionein
★ Bioaccumulation
★ Biosoprtion

論文目次

碩博士論文電子檔授權書 I
國立中央大學博碩士紙本論文延後公開/下架申請書 II
國立中央大學碩士班研究生論文指導教授推薦書 III
國立中央大學碩士班研究生論文口試委員審定書 IV
致謝 V
摘要 VI
Abstract VII
目錄 VIII
圖目錄 XII
表目錄 XV
第壹章緒論 (Introduction) 1
1.1 研究背景 1
1.2 微生物生物整治 2
1.2.1生物吸收 (Biosorption) 2
1.2.2 生物累積 (Bioaccumulation) 3
1.2.3 Pseudomonas sp. A46之前人研究 4
1.3 重金屬之汙染 4
1.3.1 銅之型態、汙染來源及對人體的危害及影響 4
1.3.4 鎘之型態、汙染來源及對人體的危害及影響 5
1.4 金屬硫蛋白之特性及功能 8
1.5染色體整合之生物應用 8
第貳章實驗目的和實驗架構 10
第參章實驗材料與方法 (Materials and Methods) 12
第一節實驗材料 12
3.1.1 使用儀器與廠牌 12
3.1.2 常用藥品與試劑 14
第二節實驗方法 19
3.2.1 菌株來源、保存與繼代培養 (Pseudomonas sp. A46) 19
3.2.2 菌株來源、保存與繼代培養 (E. coli) 20
3.2.3 細菌Genomic DNA萃取 20
3.2.4 細菌RNA萃取 21
3.2.5 反轉錄作用 23
3.2.6 DNA/RNA洋菜膠電泳 24
3.2.7 聚合酶鏈鎖反應 25
3.2.8 TSS法之大腸桿菌勝任細胞備製及轉型 26
3.2.9 細菌之質體抽取及純化 28
3.2.10 限制酵素處理 29
3.2.11 DNA膠體純化 29
3.2.12 DNA接合反應 31
3.2.13 蛋白質萃取 31
3.2.14 蛋白質濃度測定 32
3.2.15 SDS-PAGE蛋白質電泳 33
3.2.16 蛋白質轉漬 34
3.2.17 西方免疫墨點法 35
3.2.18 細菌生長曲線測定 36
3.2.19 最小抑制濃度測定 36
3.2.20 重金屬敏感度測試 37
3.2.21 細菌生物累積實驗 38
3.2.22 EDTA淋洗測定金屬累積位置 39
3.2.23 建構反向篩選用剔除upp載體pUCKdupp 40
3.2.24 建構整合型MT1-EGFP表現片匣載體pUCK01 41
第肆章實驗結果 (Result) 43
4.1 建構upp反向篩選系統 43
4.2 以反向篩選系統整合MT1-EGFP表現片匣於Pseudomonas sp. A46 44
4.3 以RT-PCR分析Pseudomonas sp. M01金屬硫蛋白基因之mRNA表現 45
4.4 以西方墨點法評估MT1-EGFP蛋白質於Pseudomonas sp. M01內表現量與穩定度 45
4.5 以40次連續繼代後Pseudomonas sp. M01之基因穩定度檢驗 46
4.6 以西方墨點法及螢光顯微鏡圖比較MT1-EGFP蛋白質於Pseudomonas sp. M01及pJBME內表現量與穩定性之差異 46
4.7 檢驗Pseudomonas spp.對重金屬之最低抑制生長濃度(MIC)及重金屬敏感度 47
4.8 以生物固定法測試Pseudomonas sp. M01及pJBME銅及鎘之生物累積能力 47
4.9 以EDTA淋洗法分析重金屬銅及鎘之菌體結合位置 48
第伍章討論 ( Discussion ) 49
5.1 金屬硫蛋白之生物累積加強法 49
5.2 金屬硫蛋白之持續表現型片匣設計以及互換機制討論 49
5.3 染色體整合表現MT1-EGFP之表現以及未來發展 51
5.4 Pseudomonas sp. A46之生物累積以及生物吸附機制探討 52
第陸章結論 (Conclusion) 55
參考文獻 (Reference) 56
圖表 65
附加資料(Supplementary File) 83

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指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2018-7-16

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