傳統欲在大腸桿菌中表現外源蛋白，多以將帶有外源基因的質體送入來達到外源蛋白的表現，但質體存在於菌株中會對菌株造成代謝負擔並且造成菌株的不穩定。為了達到重組蛋白在大腸桿菌中的穩定表現，可將外源基因利用同源重組的方式換入大腸桿菌的染色體中，但其效率低且有基因長度的限制。本研究應用近年來新發展用於真核細胞的基因編輯系統CRISPR-Cas9，在大腸桿菌染色體產生雙股斷裂，希望提升外源基因嵌入染色體的效率。我們建構CRISPR-Cas9 系統所需質體，並在送入大腸桿菌BL21 (DE3)、MG1655 與W 後證實可切割染色體。為將抗生素表現匣嵌入染色體中的LacZ 基因裡，我們進一步結合λ-Red 蛋白recombineering 系統，證實λ-Red 蛋白在大腸桿菌中確實具有保護線性DNA的效果，並且成功利用CRISPR-Cas9 系統造成的雙股斷裂，達到增加在LacZ 基因中利用同源重組換入1.4 kb 之外源片段的效率。我們期許能夠在大腸桿菌中利用CRISPR-Cas9 系統建立良好的基因編輯系統，透過雙股斷裂來成功達到更有效率地同源重組，並且在未來能夠插入更長的外源基因片段，達到利用基因編輯來產生生質化學品的目的。

In E. coli, foreign genes can be introduced and expressed via plasmid transformation or integration into its chromosome. However, plasmid-based methods are prone to plasmid instability and unstable protein expression. In order to obtain an E. coli strain that stably expresses recombinant proteins, foreign genes can be integrated into its chromosome via homologous recombination. However, the recombination
efficiency is low and the lengths of foreign genes are limited. In this study, we aimed to use the CRISPR-Cas9 system, a gene-editing system that causes double-strand
breaks (DSBs) in E. coli, hoping to achieve more efficient homologous recombination. We constructed plasmids required for the CRISPR-Cas9 system and proved that the system caused DSBs in E. coli BL21 (DE3), MG1655, and W. To protect the linear donor DNA cassette encoding antibiotics from degradation, we also exploited the bacteriophage λ-Red proteins and demonstrated that λ-Red proteins protected linear DNA from degradation and the 1.4 kb linear template was successfully inserted into the LacZ locus. We expect that the CRISPR-Cas9 system can insert longer templates into
E. coli genome in a more precise and facile manner. The platform may facilitate the production of biofuels or biomass-derived chemicals in the future.

第一章 文獻回顧 1
1-1 大腸桿菌表現系統 1
1-1-1 外源基因在大腸桿菌中之表現 1
1-1-2 pET表現系統 2
1-2 基因編輯的發展 3
1-2-1 大腸桿菌基因編輯 3
1-2-2 現有之同源重組(homologous recombination)方法 3
1-2-3 利用雙股斷裂搭配λ-Red系統促進同源重組 5
1-2-4 大腸桿菌的自我修復機制 6
1-3 CRISPR系統 7
1-3-1 CRISPR系統的起源與組成 7
1-3-2 CRISPR系統的分類與作用機制 7
1-3-3 CRISPR-Cas9系統於基因編輯上的應用 8
1-4 研究動機 9
第二章 材料與方法 16
2-1 同源重組模板質體建構與製備 16
2-1-1 同源重組質體之建構 16
2-1-2 線性同源重組模板的製備 19
2-2 大腸桿菌中FLP/Frt 系統的建立 22
2-2-1 FLP/Frt 系統電穿孔勝任細胞製備 22
2-2-2 以colony PCR與定序驗證同源重組 24
2-2-3 利用FLP-Frt系統剔除抗生素表現匣 25
2-3 CRISPR-Cas9系統的建構與運用 26
2-3-1 pCas9與pCRISPR質體建構 26
2-3-2 利用CRISPR-Cas9系統在大腸桿菌中產生雙股斷裂 28
2-3-3 在大腸桿菌中驗證雙股斷裂的自我修復效果 29
2-4同源重組系統建立 29
2-4-1 CRISPR-Cas9/ λ-Red系統建構 29
2-4-2 利用CRISPR-Cas9/λ-Red 系統促進在大腸桿菌中之同源重組 30
2-5 驗證大腸桿菌中的同源重組 31
第三章 實驗結果 40
3-1 CRISPR-Cas9 系統建構 40
3-2 CRISPR-Cas9系統在大腸桿菌BL21 (DE3)中的切割 41
3-3 CRISPR-Cas9系統與GmR模板在BL21 (DE3)菌株中之同源重組 42
3-4 不同抗生素在不同大腸桿菌菌株中的同源重組效率 44
3-5 CRISPR-Cas9 系統在大腸桿菌MG1655中的切割與自我修復機制 45
3-6 CRISPR-Cas9系統與TcR模板在MG1655菌株中之同源重組 46
3-7 CRISPR-Cas9系統在不同菌株中的切割與同源重組 47
第四章 討論 63
第五章 未來展望 66
5-1 建立可重複利用型CRISPR-Cas9系統 66
5-2 利用CRISPR-Cas9系統幫助生產1,4-丁二醇 67
5-3 利用CIChE方法增加外源基因複本數 67
第六章 參考文獻 68

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