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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨 | |
| dc.contributor.author | Shao-Ying Hsieh | en |
| dc.contributor.author | 謝少穎 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:08:21Z | - |
| dc.date.available | 2023-01-01 | |
| dc.date.copyright | 2020-02-05 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-02-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66781 | - |
| dc.description.abstract | Pichia pastoris是一種嗜甲醇酵母菌，因其生長快速、低成本、可大量表現蛋白質，且具有在原核細胞無法進行的轉譯後修飾等特性，因此常用來大量表達異源蛋白質。而其AOX1 啟動子 (alcohol oxidase I promoter) 因具有高度調控嚴謹性，且在被甲醇誘導後可大量產生目標蛋白質，因此在P. pastoris中為最常用來表現蛋白質之啟動子。本研究希望藉由調控AOX1啟動子之轉錄因子表現量，來提昇AOX1啟動子表現效率，進而增加目標蛋白質表現量。本研究將以活化子Mxr1及抑制子Nrg1做為切入點，利用基因調降系統CRISPRi (clustered regularly interspaced palindromic repeats -based interference) 抑制其抑制子Nrg1，並用基因活化系統CRISPRa (CRISPR-based activation) 提升其活化子Mxr1，並以AOX1 啟動子表現綠色螢光蛋白質 (mEGFP) 作為評估方式，希望藉由降低抑制子Nrg1或提升活化子Mxr1表現量，進而提升AOX1 啟動子綠色螢光蛋白質表達效率。結果顯示分別以CRISPRi抑制Nrg1抑制子及以CRISPRa提升Mxr1活化子確實皆可提升AOX1 啟動子之螢光蛋白質表現量，其中Nrg1 mRNA降低為控制組之0.5倍；而Mxr1 mRNA可提升為控制組之4~6倍，也驗證CRISPRi與CRISPRa基因調控工具於P. pastoris之可行性。此外，本研究另進行同時表達CRISPRi與額外表現Mxr1之協同實驗，且證實可使AOX1啟動子效率較單一方法之下率更高，螢光表現更大量。 | zh_TW |
| dc.description.abstract | Pichia pastoris (reclassified as Komagataella phaffii) is commonly used as expression system for heterologous proteins. The strictly-regulated and methanol inducible AOX1 promoter (PAOX1) is the most extensively used for proteins expression in P. pastoris. Several transcriptional factors are involved in the regulation of PAOX1 including the transcriptional activator, MXR1, and the repressor, NRG1. In this study, the PAOX1 efficiency was enhanced by clustered regularly interspaced palindromic repeats -based interference (CRISPRi) to downregulate Nrg1 and CRISPR-based activation (CRISPRa) to upregulate Mxr1 expression. Evaluated by the expression a monomeric enhanced green fluorescent protein (mEGFP) gene, the Nrg1 knockdown by CRISPRi successfully blocked the RNA polymerase initiation or elongation while transcription. In addition, the activation of Mxr1 by CRISPRa recruited more transcriptional activators to enhance gene expression. Our results show that the mEGFP expression enhanced by Nrg1 knockdown reached the maximum level 1 day earlier than that of the non-Nrg1 repressed strains. Similar results were also observed in the Mxr1 activation strains by CRISPRa to extend guide RNAs to include effector protein recruitment sites, and to recruit MCP-VP64 fusion protein. Consequently, VP64 recruited more transcription activators to help transcription of Mxr1. This CRISPRi and CRISPRa gene regulation platform provides a simple approach for regulate gene expression on in P. pastoris. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:08:21Z (GMT). No. of bitstreams: 1 U0001-1801202010295600.pdf: 2584514 bytes, checksum: c1f005103a274fd2d5aa0796132d7fd3 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 摘要 I Abstract II 目錄 III 圖目錄 VI 表目錄 VII 第一章 前言 1 一、異源表達系統 1 二、Pichia pastoris 嗜甲醇酵母菌表現系統 2 1. 嗜甲醇酵母菌 Pichia pastoris 甲醇代謝路徑 2 2. P. pastoris 蛋白質表現系統 3 三、提升 P. pastoris 表現量 7 1. 生產菌株優化及篩選 7 2. 發酵條件優化 8 四、AOX1 啟動子調控及其轉錄因子 9 1. 活化子 Mxr1、Prm1、Mit1 10 2. 抑制子 Nrg1、Mig1、Mig2 11 五、基因調控系統 12 1. CRISPR (clustered regularly interspaced short palindromic repeat) 12 2. CRISPRi (CRISPR interference) 12 3. CRISPRa (CRISPR activation) 13 六、研究動機 15 1. 目的 16 2. 轉錄調控策略 16 3. 目標 17 七、預期結果 20 1. 以 CRISPRi 抑制 Nrg1 20 1.1 抑制綠色螢光蛋白生產菌株 E3 之 Nrg1 表現量 20 1.2 抑制額外表現 Mxr1 綠色螢光蛋白生產菌株 E3M1 之 Nrg1 表現量 20 2. 以 CRISPRa 提升提升綠色螢光蛋白生產菌株 E3 之 Mxr1 表現量 20 八、本論文菌株代號說明 22 第二章 材料與方法 23 一、實驗菌株與培養條件 23 1. 細菌 23 2. 真菌 23 3. 菌株保存 23 二、培養基 24 三、表現載體建構 26 1. pAOX2K-dCas9-pU6-sgNrg1 27 2. pAOX2H-dCas9-pU6-sgNrg1 27 3. pAOX2K-dCas9-pU6-scMxr1 27 4. pAOX2HH-MCP-VP64 28 四、嗜甲醇酵母菌電穿孔轉形 33 1. P. pastoris 勝任細胞製備 33 2. 電穿孔轉形 33 五、嗜甲醇酵母菌染色體 DNA 分析 34 1. 轉形株染色體分析 34 六、轉形株培養與分析 35 1. 試管誘導 35 2. 搖瓶誘導 35 七、mRNA 表現量分析 36 八、蛋白質分析 38 1. 螢光強度分析 38 2. 胞內蛋白質萃取 38 3. 聚丙烯醯胺膠體電泳分析 38 4. 西方墨點法 39 5. AOX 活性分析 40 第三章 結果 44 一、轉形菌之建立與表現載體轉入之分析 44 1. 建立 CRISPRi 抑制 Nrg1 之胞內型綠色螢光蛋白質生產菌株 44 1.1 胞內型綠色螢光蛋白質生產菌株 44 1.2 建立 CRISPRi 抑制 Nrg1 之胞內型綠色螢光蛋白質生產菌株 44 2. 建立 CRISPRi 抑制 Nrg1 並額外表現 Mxr1 之胞內型綠色螢光蛋白質菌株 45 2.1 額外表現 Mxr1 之胞內型綠色螢光蛋白質生產菌株 45 2.2 建立 CRISPRi 抑制 Nrg1 並額外表現 Mxr1 胞內型綠色螢光蛋白質菌株 45 3. 建立 CRISPRa 提升 Mxr1 之胞內型綠色螢光蛋白質生產菌株 46 二、CRISPRi 抑制轉錄因子 Nrg1 對異源蛋白質生產的影響 53 1. 抑制 Nrg1 加強胞內型綠色螢光蛋白質的生產 53 2. 不同 sgRNA 位置具有不同螢光提升效果 53 3. 以 CRISPRi 抑制 Nrg1 對其轉錄量之影響 54 4. 抑制 Nrg1 提升 AOX1 啟動子轉錄效率 54 5. 抑制 Nrg1 對額外表現 Mxr1 之胞內型綠色螢光蛋白質的生產影響 55 三、CRISPRa 提升轉錄因子 Mxr1 對異源蛋白質生產的影響 67 1. 提升 Mxr1 加強胞內型綠色螢光蛋白質的生產 67 1.1 E3aM1~E3aM6 螢光蛋白質表現變化 67 1.2 E3aM1V~E3aM6V 螢光蛋白質表現變化 68 2. 以 CRISPRa 提升 Mxr1 對其轉錄量之影響 68 3. 提升 Mxr1 可提升 AOX1 啟動子轉錄效率 69 第四章 討論 75 一、抑制 Nrg1 對提升 AOX1 啟動子表現量之探討 75 二、提升 Nrg1 對提升 AOX1 啟動子表現量之探討 76 1. CRISPRa 提升 Mxr1 活化子之能力 76 2. CRISPRa 提升 Mxr1 與直接額外表達 Mxr1 比較 77 第五章 結論 79 第六章 未來展望 80 第七章 參考文獻 81 | |
| dc.language.iso | zh-TW | |
| dc.subject | Pichia pastoris | zh_TW |
| dc.subject | gene regulation | zh_TW |
| dc.subject | CRISPRi | zh_TW |
| dc.subject | CRISPRa | zh_TW |
| dc.subject | Nrg1 | zh_TW |
| dc.subject | Mxr1 | zh_TW |
| dc.subject | Mxr1 | en |
| dc.subject | Pichia pastoris | en |
| dc.subject | methanol | en |
| dc.subject | AOX1 promoter | en |
| dc.subject | transcription factor | en |
| dc.subject | Nrg1 | en |
| dc.title | 以CRISPRi及CRISPRa策略加強Komagataella phaffii (Pichia pastoris) AOX1啟動子效率 | zh_TW |
| dc.title | Enhancing AOX1 promoter efficiency of Komagataella phaffii (Pichia pastoris) using CRISPRi and CRISPRa | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 凌嘉鴻,陳浩仁,張世宗,林晉玄 | |
| dc.subject.keyword | Pichia pastoris,gene regulation,CRISPRi,CRISPRa,Nrg1,Mxr1, | zh_TW |
| dc.subject.keyword | Pichia pastoris,methanol,AOX1 promoter,transcription factor,Nrg1,Mxr1, | en |
| dc.relation.page | 86 | |
| dc.identifier.doi | 10.6342/NTU202000185 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-02-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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