隨著氣候變遷以及能源危機的驅動下，已有許多生質能利用微生物結合基因工程大規模的被生產出來。其中藍綠菌是被認為近年來最具有潛力的微生物之一，能進行光合作用將二氧化碳轉換成可用的生質能，不需提供碳水化合物來當作碳源。然而，現有的基因工程技術是利用同源重組的方法來剔除目標基因，進而調控代謝路徑。但由於藍綠菌為多倍體生物，利用傳統方法並不易將目標基因完全剔除。而新興的CRISPRi (CRISPR interference)基因調控系統，僅需設計sgRNA (single guide RNA)來辨視目標基因即可達到抑制基因表現的功效，它具有設計便捷，且能同時調控多重目標基因等優勢。本研究首先在藍綠菌-細長聚球藻PCC 7942中以黃螢光蛋白(EYFP)作為報導基因來探討多種不同啟動子之表現強度。接著，結合前述之啟動子(如conII啟動子)我們建立一套CRISPRi基因調控系統，並先以黃螢光蛋白基因的抑制程度來驗證CRISPRi系統的效用。從螢光顯微鏡中可觀察到螢光表現明顯的被抑制，不論是sgRNA辨視的位置為conII啟動子或eyfp基因，而在流式細胞儀分析可發現到螢光表現不同程度的抑制效果(72-99%)，其中我們發現當sgRNA標的至eyfp基因之位置越接近轉錄起始點時其抑制效果越好。之後，我們進行21天長期培養來觀察CRISPRi系統的穩定性以及細胞生長曲線，我們發現到CRISPRi系統能長期穩定抑制基因表現並且不會對細胞生長造成負面影響。接著，我們進一步利用CRISPRi系統來抑制glgc、sdhA或sdhB等內源基因表現，並在缺乏氮源的情況下培養，藉此改變PCC7942代謝路徑來促進琥珀酸之產量。由mRNA分析結果同樣發現，標的基因位置越接近轉錄起始點時，效果越好，且各組之琥珀酸產量也隨著基因抑制效果的增加而提升，最高可達到0.63 mg/L，為wild type的12倍。本研究證實CRISPRi系統可應用於代謝工程中藉此來優化目標產物產量。

Genetically engineered cyanobacteria hold promise as a host organism for bio-derived product production, but traditional method to modulate cyanobacterial metabolism requires time-consuming gene knock-in/knock-out. For gene suppression, a novel RNA-guided CRISPR interference (CRISPRi) technology was recently developed, which only requires co-expression of dead Cas9 protein (dCas9) and single guide RNA (sgRNA) that target specific genomic sequences without permanently altering genomic DNA. To explore CRISPRi for the modulation of cyanobacterial gene expression and succinate production, we first compared the strength of various promoters in S. elongates PCC7942 using EYFP as the reporter. Next, we developed an CRISPRi system in PCC7942 by co-expressing dCas9 (driven by the smt promoter) and sgRNA (driven by J23119 promoter) to repress the expression of EYFP (driven by conII promoter). Fluorescence microscopy confirmed significant downregulation of EYFP, either by sgRNA targeting to the conII promoter or eyfp gene. Using different sgRNAs targeting different gene regions, CRISPRi suppressed EYFP expression with efficiencies ranging from 72% to 99%. In addition, repression was inversely correlated with the target distance from the transcription start site. The CRISPRi-mediated gene suppression caused no appreciable side effects and persisted for 21 days. Using CRISPRi, we successfully knocked down the expression of endogenous genes such as glgc, sdhA, sdhB, which allowed us to modulate the metabolic flux and enhanced the succinate production to 0.63 mg/L under nitrogen deprived conditions in S. elongates PCC7942. Similarly, repression was inversely correlated with the target distance from the transcription start site and consistent with the succinate production. This study paved a new avenue to regulate the metabolic pathways of PCC7942 by CRISPRi for optimal production of desired bio-derived products.

摘要 I
Abstract II
目錄 III
圖目錄 VII
表目錄 X
第一章 文獻回顧 1
1-1藍綠菌S. elongatus PCC7942簡介 1
1-2 RNA調控機制 2
1-3 CRISPR基因編輯與調控系統 4
1-3-1 CRISPR/Cas9基因剪輯系統 4
1-3-2 CRISPRi基因調控系統 5
1-4 琥珀酸介紹 7
1-5 研究動機 10
第二章 實驗材料及方法 17
2-1建構質體方法 17
2-1-1 DNA引子黏合反應(primer annealing) 17
2-1-2 PCR反應 17
2-1-3 線性DNA磷酸化反應(phosphorylation reaction) 18
2-1-4限制酶(DNA restriction enzyme)反應及接合酶(DNA ligase)反應 18
2-1-5反轉錄(Reverse transcription)合成單股cNDA 18
2-2質體建構:不同啟動子與RBS之螢光表現系統 19
2-2-1 不同啟動子螢光表現系統之建構 19
2-2-2 不同RBS之建構 22
2-3 CRISPRi基因調控系統所需質體之建構 23
2-3-1 CRISPRi基因調控系統抑制黃螢光蛋白測試模型質體之建構 23
2-3-2 CRISPRi基因調控系統抑制藍綠菌內源性基因所需質體之建構 25
2-4 Synechococcus elongatus PCC 7942培養條件 27
2-5 Synechococcus elongatus PCC 7942質體轉型 28
2-5-1質體轉型 28
2-5-2以colony PCR與定序驗證同源重組至染色體正確位置 28
2-6 流式細胞儀分析(flow cytometry) 29
2-7螢光顯微鏡分析(Fluorescence microscopy analysis) 30
2-8即時偵測同步定量反轉錄聚合酶連鎖反應(qRT-PCR) 31
2-8-1 抽取細胞mRNA與反轉錄合成單股cDNA 31
2-9 胞內肝醣定量分析(Quantification of glycogen) 33
2-10 琥珀酸產量分析(Succinate analysis) 34
第三章 實驗結果與討論 49
3-1不同啟動子之螢光表現系統 49
3-1-1不同啟動子之螢光表現系統之建立 49
3-1-2不同啟動子之螢光表現系統之分析 49
3-2 CRISPRi基因調控系統 50
3-2-1 建立CRISPRi基因調控系統以及黃螢光測試模型 51
3-2-2 CRISPRi基因調控系統之抑制成效及其穩定性與毒性之分析 52
3-2-3 改良CRISPRi基因調控系統之誘導表現 55
3-2-4 建立CRISPRi基因調控系統來抑制藍綠菌PCC7942內源性基因表現 56
3-2-5 CRISPRi系統抑制藍綠菌內源性基因表現之成效以及琥珀酸產量之分析 58
3-3 結論 59
第四章 未來工作 73
參考文獻 78

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