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研究生: 許泰銘
Hsu, Tai-Ming
論文名稱: 大腸桿菌多套數質體的分離機制
Partition Mechanism of High-Copy Number Plasmids in Escherichia coli
指導教授: 張宜仁
Chang, Yi-Ren
學位類別: 碩士
Master
系所名稱: 物理學系
Department of Physics
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 67
中文關鍵詞: 多套數質體質體分離機制
英文關鍵詞: high-copy number plasmids, plasmid partition mechanism
DOI URL: http://doi.org/10.6345/THE.NTNU.DP.005.2018.B04
論文種類: 學術論文
相關次數: 點閱:147下載:3
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  • 多套數質體不具有類似低套數質體的主動分離機制,其分離機制目前仍不明確。多套數質體會在細菌細胞內特定位置形成質體群聚。細胞分裂過程質體群聚裂成兩個小群聚,分裂的小群聚移動遠離原有群聚的位置,將質體傳遞至子代細胞。對於這樣的多套數質體群聚與分離過程,我們採取兩種實驗觀察方式:細胞分裂多套數質體數量變化與多套數質體群聚內單一質體運動。
    首先,細胞多套數質體數量觀察,我們設計測量兩種具有相同複製原點、不同螢光抑制操作系統(fluorescent repressor-operator systems, FROS)標記之 ColE1衍生質體,轉形於同一菌株,統計細胞分裂親代細胞與子代細胞質體數量。發現ColE1衍生質體之間的質體不相容,以及證實細胞分裂過程質體分配具有子代分配傾向。
    質體群聚內的單一質體追蹤,我們突變ColE1複製原點,利用抑制子-啟動子配對(repressor-promoter pair) PhlF與PphlF ,調控RNAII的表達,控制質體複製數量。藉此限定質體群聚當中一種螢光色標記質體為單一質體,以螢光顏色區分,追蹤質體群內的單一質體動態。單一質體追蹤軌跡迴轉半徑分析證實細胞內多套數質體存在兩種不同的運動,我們分別稱為質體侷限運動與跳躍運動。兩種運動的平均方均根位移(mean square displacement, MSD) 曲線、擴散係數與質體位置重疊指數,表示單一質體運動受到質體群聚的質體數量影響。我們也同時應用HupA的全細胞螢光顯微術(Whole-cell fluorescence microscopy),觀察細胞類核空間變化與質體運動的關聯。其結果表示單一質體擴散運動受到類核佔據空間排擠。
    綜合本次實驗結果,我們認為細胞內多套數質體的空間分布包含漂散的多套數質體與質體群聚。漂散的多套數質體分布於類核外空間進行擴散運動。細胞分裂過程類核空間結構改變,漂散的多套數質體從新的類核外空間進行長距離移動,藉此達成多套數質體分離。

    High-copy number plasmids typically present as multiple clusters at specific cellular region in bacterial cells, even during their replication and partition. However, unlike the discovered active partition system in low-copy plasmids, their essential partition mechanism is still unclear. We approach this problem in investigation of the variation of inherited plasmid copy number and single plasmid motion in clusters in bacterial cell.
    We co-transformed two ColE1 derivative plasmid with different fluorescent repressor-operator systems and antibiotic resistance gene, pLacOIC2.bx and pTetORK34.b, into E. coli strain BW25113. The distributions and motions of the plasmids are investigated respectively at single cell level. Our results show that the nature of competition between plasmids and the variance of the plasmid inheritance ratio for both of two plasmids from parent to daughter cells. The inheritance ratio showing more diverse distribution than simple binomial distribution. Additionally, We co-transformed the plasmid pLacOIC2.bx and pTetORK34.bp, modified from pTetORK34.b, with the synthetic replication origin into BW25113. Based on the plasmid pTetORK34.bp copy number reduction via the repression of replication primer RNAII expression which is regulated by genetic circuit of PhlF and PphlF, it provides the way to efficiently track a single plasmid motion in the plasmid clusters. According to radius of gyration of the single plasmid traces, these single plasmid motions distinguished into jump-like motion and confined motion. For the traces of these two motion, we calculated average mean square displacement, diffusivity and degree of co-localization. Our results suggest that plasmid motion determined by the plasmid clusters. Furthermore, we monitored the single plasmid motion and spatial distribution of nucleoids labeled by HupA-mTurquoise2 within a cell cycle. Our results show that the single plasmid motion was excluded by the nucleoid.
    The heterogeneity of partition is revealed by the variation of plasmid copy number and single plasmid motions in clusters. High-copy number plasmids are organized as both clusters and single random copies inside bacteria. We believe that partition mechanism is dominated by spatial distribution of nucleoids.

    摘要 I Abstract II 致謝 IV 目錄 V 圖目錄 VIII 表目錄 IX 第一章 緒論 1 1.1 質體介紹 1 1.1.1 質體基本性質 1 1.1.2 質體維持 2 1.2 多套數質體分離機制 3 1.2.1 隨機分布假說 3 1.2.2 質體群聚與替代假說 3 1.2.3 類核排擠模型 5 1.2.4 混合分布模型 6 1.3 研究目標與架構 6 第二章 實驗設計原理 8 2.1 質體螢光標記-FROS 9 2.2 複製原點ColE1工作機制與應用 10 2.3 類核螢光標記 11 2.4 單分子追蹤 12 第三章 實驗材料與方法 18 3.1 質體 18 3.1.1 pTetORK34.b與pTetORK34.bp 19 3.1.2 pLacOIC2.bx 20 3.1.3 pZC320-tetO.b與pZC320-lacO.b 20 3.1.4 p15AA-phlFH.tq 20 3.2 菌株 21 3.3 光學儀器 22 3.4 實驗操作與分析 22 3.4.1 單一質體螢光標記亮度分布 22 3.4.2 細胞內多套數質體數量分佈 23 3.4.3 細胞內質體數量控制 24 3.4.3.1 菌株BWP3 24 3.4.3.2 菌株BWP23 25 3.4.4 質體群聚下的單一質體運動追蹤 26 第四章 實驗結果與討論 32 4.1 單一質體螢光標記亮度分布 32 4.2 細胞內多套數質體數量分佈 33 4.2.1 細胞內質體數量的組成 33 4.2.2 子代細胞質體繼承 34 4.2.3 細胞分裂質體分配統計 35 4.3 細胞內質體數量控制 36 4.4 質體群聚下的單一質體運動追蹤 37 4.4.1 質體軌跡迴轉半徑 38 4.4.2 質體擴散運動與質體群聚 38 4.4.3 質體運動與細胞類核變化 39 第五章 結論 54 參考文獻 55 附錄 58 附錄1. 質體建構引子表 58 附錄2. 質體基因圖譜 62 附錄3. 螢光標記多套數質體數量計算 63 附錄4. MATLAB程式碼 66

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