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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李佳音(Chia-Yin Lee) | |
dc.contributor.author | YI-Han Hsieh | en |
dc.contributor.author | 謝依涵 | zh_TW |
dc.date.accessioned | 2021-06-13T00:26:29Z | - |
dc.date.available | 2007-07-30 | |
dc.date.copyright | 2007-07-30 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-25 | |
dc.identifier.citation | 蘇訓正。2002。Amycolatopsis azurea之N-醯化胺基酸消
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28861 | - |
dc.description.abstract | 土壤中之微生物具有高度多樣性以及含有許多未知待開發之產物,因此,利用建構好之土壤多源基因庫來開發新型酵素、化合物、抗生素或是菌種,已經是今日的趨勢。本篇研究主要擬利用本研究室已建構好之土壤多源基因庫篩選目標產物,利用功能性篩選以及基因序列篩選方法進行。本篇研究第一部分為檢驗前人利用聚合酶連鎖反應方式,由土壤多源基因庫篩選出來的ORF4基因序列,疑似具有N-醯化胺基酸消旋酶,經過次選殖及酵素呈色法試驗,結果,證實此疑似具有N-醯化胺基酸消旋酶活性之基因片段無法表現該酵素活性。第二部份為利用前人建構的土壤多源基因庫,以pH 8.5的脫脂牛乳瓊脂培養基進行功能性篩選鹼性蛋白酶,同時亦利用DNA雜交方法篩選蛋白酶。經DNA雜交實驗及培養基篩選的結果並未能成功篩選出具有鹼性蛋白酶活性之菌株。將這批土壤多源基因庫抽取其BAC質體經限制酶截切後,以電泳分析發現此土壤多源基因庫具有高歧異度,仍可供日後篩選其他目標產物之用。 | zh_TW |
dc.description.abstract | Since environmental microorganisms holds a great of diversity and are available for developing multiple unknown products, environmental metagenomes can be also applied in different fields, such as medical and chemical industries nowadays. Metagenome is thought as a powerful tool to exploit chemical molecules, antibiotics, enzymes, and bacterial species. Screening of soil-based libraries can be based either on metabolic activity (function-based approach) or on nucleotide sequence (sequence-based approach). There are two sections in this study. First part, a putative N-acylamino acid racemase naaar gene, orf4 gene are amplified by PCR and cloned into E. coli BL21(DE3). Analysis of the cell extracts from this clone, the NAAAR activity was unable observed. Second part, the alkaline serine protease was screened by using this soil metagenome library. Both of function-based screening, that was assayed on pH 8.5 skim milk plates, and DNA-DNA hybridization. It didn’t succeed to screen out bacteria with alkaline serine protease through function-based and sequence-based screenings. After extraction and digestion of plasmids from the soil metagenome library, it was found that this metagenome library provides a great of diversity for screening other target products in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T00:26:29Z (GMT). No. of bitstreams: 1 ntu-96-R93623021-1.pdf: 2829603 bytes, checksum: e3bd9ab94196d387468070a61bbb9414 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 目錄
頁次 中文摘要 i 英文摘要 iii 目錄 v 表次 xi 圖次 xii 壹、前言 一、微生物多源基因體 (Metagenome)之相關研究 1 1. 環境微生物之多樣性 1 2. 微生物多源基因庫之簡介 2 3. 微生物多源基因庫之應用 3 二、微生物多源基因庫之篩選 5 1. 功能性篩選 (Function-based screening)目標產物 6 2. 基因序列性篩選 (Sequence-based screening)目標產物 7 三、N-醯化胺基酸消旋酶之相關研究 8 四、鹼性蛋白酶之相關研究 10 五、研究緣起與目的 12 貳、材料與方法 І. 實驗材料 14 一、實驗菌株與質體 14 二、培養基 14 三、藥品與試劑 17 四、實驗中使用之套組 19 五、各種溶液及緩衝溶液 19 1. 抽取質體所需溶液 19 2. 雜交所需緩衝溶液與試劑 19 六、儀器 20 七、其他 22 Ⅱ. 實驗方法 22 一、由土壤多源基因庫選殖出pBAB4F2質體上 之ORF4 N-醯化胺基酸消旋酶活性測試 22 1. 利用聚合酶連鎖反應放大pBAB4F2上之 ORF4 DNA片段(命名為orf4) 22 2. 接合作用 23 3. 熱休克轉型作用 24 4. 轉型株挑選 25 二、 pET-orf4選殖株之N-醯化胺基酸 消旋酶活性測試 26 1. N-醯化胺基酸消旋酶粗酵素液製備 26 2. N-醯化胺基酸消旋酶酵素活性分析 27 三、 功能性篩選土壤多源基因庫中之鹼性蛋白酶 28 1. 活化土壤多源基因庫 28 2. 利用鹼性脫脂牛乳瓊脂培養基篩選 28 3. 抽取土壤多源基因庫中之BAC質體 29 4. 限制酶截切以及DNA純化 30 5. 電衝法轉型細胞製備及電衝法轉型作用 30 四、 基因序列篩選土壤多源基因庫中之鹼性蛋白酶 31 1. DIG(Didoxigenin)探針標定之合成 31 2. DNA漬片製備 33 3. 雜交反應 35 4. 嚴苛度漂洗 36 5. 免疫偵測法 36 6. 脫色處理 37 7. 去探針處理 37 參、結果 一、 pBAB4F2 之N-醯化胺基酸消旋酶活性測定 38 1. pBAB4F2上之orf4基因選殖及建構重組質體 38 2. orf4基因序列定序與資料庫中的基因序列比對分析 39 3. N-醯化胺基酸消旋酶粗酵素液之活性測試 39 二、 土壤多源基因庫以功能性方式篩選鹼性蛋白酶 40 1. 脫脂牛乳瓊脂培養基篩選鹼性蛋白酶 40 2. 土壤多源基因庫之歧異度 42 三、基因序列篩選土壤多源基因庫之鹼性蛋白酶 43 1. 設計鹼性絲胺酸蛋白酶基因探針 43 2. DNA雜交反應篩選鹼性絲胺酸蛋白酶基因分析 43 肆、討論 1. pBAB4F2-19 之N-醯化胺基酸消旋酶活性測定 45 2. 功能性篩選土壤多源基因庫 45 3. 多源基因庫建構方式及應用之探討 46 伍、結論 一、重組質體pET-orf4 之N-醯化胺基酸消旋酶活性檢測 48 二、以功能性篩選土壤微生物基因庫中的鹼性蛋白酶 48 三、以序列篩選方式多源基因庫 49 陸、參考文獻 50 表次 表一、本研究所使用之引子 69 表二、本篇研究所使用的菌株與質體 70 表三、orf4基因序列分析 71 圖次 圖一、以pBAB4F2為模板選殖出orf4所使用的 PCR引子組合、及PCR產物片段大小 73 圖二、pET-orf4表現載體建構示意圖 74 圖三、經由PCR選殖出的orf4片段之電泳圖 75 圖四、經由1.4.3.DNA extraction kit純化後的orf4 76 圖五、經由限制酶EcoRІ、HindШ 截切確認黏合作用結果之電泳圖 77 圖六、使用degenerate primer abFP1a/npRP2a 引子組進行colony PCR 78 圖七、orf4核酸及胺基酸序列 79 圖八、pET-orf4重組質體在28℃下的誘導表現情形 82 圖九、重組質體 pET-orf4 在不同時間點,28℃下活性測試 83 圖十、重組質體 pET-orf4在不同 時間點下酵素活性測試及標準曲線 84 圖十一、使用pH 8.5脫脂牛乳培養基初步篩選鹼性蛋白酶 85 圖十二、將初步篩選出現溶解環之菌株質體 轉型送入E. coli XL1Blue後塗抹在pH 8.5之脫脂 牛乳瓊脂培養基上後出現之溶解環 87 圖十三、自土壤多源基因庫菌株中抽取質體來 觀察此基因庫基因歧異度 88 圖十四、使用限制酶EcoRІ、HindⅢ截切由 土壤多源基因庫得到質體之電泳分析圖 91 圖十五、根據腸炎弧菌 No. 93 prtA基因設計之探針 94 圖十六、使用腸炎弧菌No. 93之染色體為模板 製備絲胺酸蛋白酶探針 95 | |
dc.language.iso | zh-TW | |
dc.title | 利用土壤多源基因庫篩選新穎酵素之研究 | zh_TW |
dc.title | The study of using metagenome library to screen novel biocatalysts | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳惠芬,陳昭螢 | |
dc.subject.keyword | 土壤多源基因庫,鹼性蛋白酶,N-醯化胺基酸消旋酶, | zh_TW |
dc.subject.keyword | soil metagenome library,alkaline serine protease,N-acylamino acid racemase, | en |
dc.relation.page | 95 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-26 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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