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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
dc.contributor.author | Pei-Hsuan Shen | en |
dc.contributor.author | 沈佩璇 | zh_TW |
dc.date.accessioned | 2021-06-15T00:14:29Z | - |
dc.date.available | 2011-06-30 | |
dc.date.copyright | 2009-06-30 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-06-26 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41225 | - |
dc.description.abstract | 建構表現載體時,抗藥性基因是最常被應用的篩選標記。近年來異源表達系統蓬勃發展,除了一般基因重組的微生物,其他如基因改造植物(Genetically modified plants, GMP),也常以抗生素抗性基因當作標示基因,其所攜帶的轉殖基因可能經由花粉傳播,或在土壤造成微生物間基因水平移轉(Horizontal gene transfer)的問題。同時,由於人類過分依賴抗生素,衍生成抗生素濫用情形,造成抗藥性菌株增加,未來許多疾病可能會造成無藥可醫的窘境。因此許多先進國家已嚴格限制,甚至全面禁止抗生素的使用。所以如果能尋找新的替代標示基因來取代抗生素基因,將可解決此問題的發生。
本研究從耐輻射球菌中選殖超氧歧化酶(sod)基因,此基因廣泛存在於好氧生物體中,可催化活性氧物質超氧陰離子,將其歧化成過氧化氫和氧氣。將此sod基因片段建構在分子選殖中常使用的大腸桿菌表達載體pET系統中。再將此載體轉形至E. coli中,利用一種可生成超氧陰離子的化合物 ─ methyl viologen,配合一帶正電性物質CX (Compound X)來篩選出具有sod基因載體的轉形株。進行固態培養基、液態培養基,及同時固態和液態條件的篩選,並模擬轉形條件及實際轉形實驗測試,結果顯示在特定菌數下,可經由液態和固態培養基篩選出帶有sod質體菌株。利用此篩選方式顯示sod基因可作為一篩選標記替代抗藥性基因,可避免使用抗生素。 | zh_TW |
dc.description.abstract | To construct expression vector, antibiotic resistance gene is the most used selectable marker. Recently, the heterologous expression system has been developed extraordinarily. The antibiotic resistance gene is often useful in enabling not only genetic recombination microorganism but also genetically modified plants (GMP). However, there are some problems about widespread occurrence of transgenes in ecosystems dispersed through pollen or horizontal gene transfer in the soil microorganism. Meanwhile, because human rely on antibiotics excessively , it is in increasing antibiotics resistance strains that the misuse of antibiotics phenomenon results. The serious effect will lead no drugs to treat disease in the future. For this reason, many developed countries have limited or even prohibited the use of antibiotics. It is necessary to look for a new selectable gene to replace for antibiotic resistance gene.
The goal of this study is to establish a plasmid with sod gene which is cloned from Deinococcus radiodurans. All of aerobic organism have the sod gene whose protein product can catalyze superoxide anion to hydrogen peroxide and oxygen. First, the sod gene was inserted into a E. coli expression vector- pET and then the pET-SOD vector was transformed into E. coli. Methyl viologen which produced superoxide anion and a positive chemical CX was used to screen whether transformant had sod gene vectors. The method of screen included solid medium only, broth medium only and both solid and broth medium. The result showed that E. coli with sod plasmid could be selected in specific cell numbers through both solid and broth medium screen. This method can be a new approach to replacement for antibiotic resistance gene. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T00:14:29Z (GMT). No. of bitstreams: 1 ntu-98-R96b47404-1.pdf: 2357809 bytes, checksum: 270bfc140f284ef64ed9ee34dbb0c0f2 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 中文摘要 i
Abstract ii 目錄 iii 表目錄 v 圖目錄 vi 第一章 前言 1 1. 遺傳工程與異源表達 1 2. 抗生素的發展 2 3. 抗生素面臨的瓶頸 3 4. 抗藥性篩選標記及隱憂 3 5. 超氧歧化酶緒論 7 5.1 超氧自由基 7 5.2 超氧歧化酶 7 5.3 超氧歧化酶相關研究 8 6. 耐輻射球菌 9 7. Compound X的應用 9 8. 研究目的 12 第二章 材料與方法 15 一.實驗材料 15 1.菌種 15 2.質體 15 3.引子 16 4.氧化壓力試劑 17 5. Compound X配製 17 二.實驗方法 23 1. 質體之建構 23 2. SOD活性鑑定 30 3. 氧化壓力試劑篩選 32 4. 目標SOD基因確認 34 5. 菌體混濁度測定 34 第三章 結果 35 一. sod基因釣取 35 1. 耐輻射球菌sod基因 35 2. 大腸桿菌sod基因 35 二. 表現質體建構 35 1. 啟動子及sod基因重疊PCR 35 2. 啟動子 + sod基因和終止序列之黏合反應 36 3. 表現載體pET21a-P+SOD+T建構 42 4. 表現質體確認 42 三. SOD活性鑑定 46 1. 大腸桿菌MV培養 46 2. SOD蛋白質粗萃取結果 46 3. SOD電泳結果圖 46 四. 氧化壓力試劑篩選 47 1. 最小抑制濃度(Minimum Inhibitory Concentration, MIC) 47 2. 固態培養基篩選 47 3. 液態培養基篩選 48 4. 液態和固態培養基共同篩選 48 第四章 討論 59 一. 基因轉殖植物使用之篩選標記基因 59 二. sod基因篩選作用 60 1.啟動子(promoter)選擇 60 2.培養基差異 60 3.菌數影響 60 第五章 結論與未來展望 62 參考文獻 63 | |
dc.language.iso | zh-TW | |
dc.title | 以超氧歧化酶基因作為分子篩選標誌之探討 | zh_TW |
dc.title | Transformant selection using superoxide dismutase (SOD) gene | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許瑞祥,陳進庭,蔡翠敏 | |
dc.subject.keyword | 超氧歧化酶,超氧陰離子,methyl viologen,耐輻射球菌, | zh_TW |
dc.subject.keyword | superoxide dismutase,selectable marker,, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-06-26 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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