臺灣博碩士論文加值系統

微生物在金屬環境中常常能夠發展出抵抗這些有毒元素的機制，藉由從環境分離培養篩選出Pseudomonas sp. TeU，對於重金屬鎘及類金屬碲具有相當之耐受性，且具有還原碲之能力之菌株。在先前實驗中，利用跳躍子突變法我們得到一株較不具碲還原能力之突變株 (strain AU08)以及一株對鎘不具耐受性的突變株 (strain BU21)，並且鑑定出此兩株突變株被跳耀子Tn5破壞之基因分別為hflC以及金屬胜肽酶(metallopeptidase)之基因。本研究進一步將此菌株完整之hflC與metallopeptidase基因選殖並定序，進行蛋白質表現；並探討金屬胜肽酶對重金屬鎘耐受性之測試；另外以穿透式電子顯微鏡(TEM)觀察菌體與類金屬碲之作用累積情形。

A Pseudomonas strain TeU resistant to tellurite (TeO32-) and cadmium (Cd2+) ions was isolated from heavy-metal contaminated sediments by enrichment. Transposon mutagenesis of strain TeU resulted in mutants exhibiting Cd2+ sensitivity (Strain BU21) and one with decreased ability to reduce tellurite (strain AU08). Genes encoding an HflKC complex and an putative metallopeptidase were identified to be associated with the bacterium’s ability for tellurite reduction and cadmium resistance, respectively. We have cloned and sequenced the hflC and metallopeptidase gene of strain TeU. Metallopeptidase gene of strain TeU was cloned and expressed in E. coli in order to investigate if the protein could improve the bacterium’s ability to resist cadmium.Transmission electron microcopy was also employed for to observe metal precipoitation when strain TeU grown in the medium containing tellurite.

摘要 i
Abstract ii
致謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章 文獻回顧 1
1.1前言 1
1.2假單胞菌(Pseudomonas sp.)介紹 2
1.3微生物抗重金屬之生理調控 3
1.3.1生物吸附(Biosorption) 3
1.3.2金屬離子之主動運輸(Active transport of metal ions) 4
1.3.3生物累積(Bioaccumulation) 5
1.3.4生物轉換(Biotransformation) 5
1.3.5生物濾取(bioleaching) 5
1.3.6生物降解(biodegradation) 6
1.3.7生物礦化作用(Biomineralization) 6
1.4微生物與金屬碲tellurite (Te)離子毒性相關之調控 6
1.4.1對細胞膜上基因ftsH、hflK、hflC與碲(Te)離子之關係 7
1.4.2金屬胜肽酶metallopeptidase對於重金屬之關係 8
1.5研究目的 8
第二章 材料與方法 10
2.1實驗流程架構 10
2.2實驗材料 11
2.2.1本實驗所使用之菌株與質體 11
2.2.2引子 11
2.2.3藥品與試劑 12
2.2.4試劑組 12
2.2.5酵素 12
2.2.6培養基成份 13
2.2.7抗生素 13
2.2.8 DNA 電泳試劑組 13
2.2.9實驗儀器 13
2.3實驗方法 14
2.3.1菌體儲存 14
2.3.2菌株培養條件及培養基配方 14
2.3.2染色體DNA之純化(Purification of chromosomal DNA) 14
2.3.4聚合酶連鎖反應(Polymerase chain reaction, PCR) 15
2.3.5瓊脂膠體電泳分析(Agarose Gel Electrophoresis) 17
2.3.6 DNA片段之膠體萃取(DNA fragment gel extraction) 17
2.3.7 DNA接合作用(DNA -Ligation) 17
2.3.8勝任細胞製備(Preparation of competent cells ) 18
2.3.9熱休克反應(heat shock) 18
2.3.10重組質體之篩選(Screening of recombinant plasmid) 19
2.3.11質體之純化(plasmid extraction) 19
2.3.12限制酵素切割(Restriction enzyme digest) 20
2.3.13序列比對 20
2.3.14蛋白質電泳(SDS-PAGE)分析 20
2.3.15生長曲線之測定 23
2.3.16穿透式電子顯微鏡(TEM)觀察 23
第三章 實驗結果與討論 26
3.1 Pseudomonas sp.TeU之染色體純化 26
3.2實驗菌株16S rDNA之鑑定 26
3.3 Pseudomonas sp.TeU與相似菌種親源關係圖 28
3.4 Pseudomonas sp.TeU與突變株生長在tellurite金屬環境下 29
3.5聚合酶連鎖反應退化性引子設計(degenerate primers) 29
3.6 Pseudomonas sp.AU08、BU21之轉位子確認 30
3.7 Pseudomonas sp.TeU 之hflC與metallopeptidase 序列確認 30
3.8 Pseudomonas sp.AU08與BU21 突變株基因插入確認 33
3.9重組基因建構hflC gene (以pBluescriptII SK為載體) 34
3.10重組基因建構metallopeptidase gene (以pBluescriptII SK為載體) 36
3.11蛋白質電泳(SDS-PAGE)分析 38
3.12轉殖株pBluescriptII SK::metallopeptidase生長狀況分析 40
3.13類金屬胞內累積結果 41
第四章結論 42
第五章參考文獻 43

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