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標題: | 在大腸桿菌細胞中構建功能不對稱性細胞分裂 Construction of functional asymmetric cell division in Escherichia coli |
作者: | Etienne D.-C. Liao 廖得健 |
指導教授: | 黃筱鈞(Hsiao-Chun Huang) |
關鍵字: | 不對稱細胞分裂和分化,PopZ,T7RNA聚合?,DivIVA,MS2, asymmetric cell division and differentiation,PopZ,split T7 RNA polymerase,DivIVA,MS2, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 過去幾十年中,不對稱細胞分裂的分子機制已被廣泛研究。然而,在複雜與冗餘的自然調控網絡下,瞭解不對稱分裂的核心架構或是設計原理非常困難。在這個研究中,我們盼利用對稱分裂的大腸桿菌做為我們的試管平台,在其上從頭重建不對稱細胞分裂和分化。新月柄桿菌是我們已知和大腸桿菌演化上相對遙遠、並能不對稱分裂與分化成兩不同子代的單細胞生物,而PopZ是在新月柄桿菌內建立整個細胞不對稱性的關鍵極化蛋白。我們構想利用PopZ作為極性樞紐、已知PopZ的作用蛋白片段作為轉接媒介,我們可以在細胞一端招集分為兩獨立片段的T7RNA聚合酶,來不對稱開啟基因表現。如此當該細胞分裂,兩個子細胞會遺傳到不同的蛋白組合,最後造成細胞分化。我們利用螢光蛋白來驗證概念,其中我們使用了能辨認細胞膜負曲率的枯草桿菌DivIVA蛋白來解決蛋白在細胞質中擴散速度太快的問題。我們已成功地看到表現蛋白的不對稱分佈,並看到在細胞分裂時,只有一個子細胞遺傳到該表現蛋白。我們正嘗試利用MS2髮夾於活細胞中看到不對稱轉錄能力,另外我們也嘗試將螢光蛋白換成β-内酰胺酶使只有一個子細胞能在抗生素壓力下生存,藉此實現功能分化。這是我們所知第一次成功重建不對稱細胞分裂,也是合成生物學的歷史上一重要里程碑。 In the past decades, the molecular mechanism of asymmetric cell division has been extensively studied. However, it is difficult to reveal the core motif, or design principle, of asymmetric cell division, given the complexity and redundancy of the natural regulatory network. In this study, with the symmetrically dividing, Gram-negative rod-shaped γ-proteobacterium Escherichia coli (E. coli) as our test tube, we aim to reconstruct asymmetric cell division and differentiation from the bottom up. We learn that in Caulobacter crescentus, a Gram-negative oligotrophic α-proteobacterium that is evolutionarily divergent from E. coli and able to divide asymmetrically to produce two functionally distinct offspring, whereas the key polarity protein that establishes the entire programs of cellular asymmetry is Polar Organizing Protein Z (PopZ). With PopZ as the polarity hub and its known interactive protein segment as the adaptor, we can reassemble the split T7 RNA polymerase (T7 RNAP) exclusively at one pole of the cell to turn on gene expression asymmetrically. When such a cell divides into two daughters, they will inherit distinct protein and lead to differentiation. With the study of fluorescent protein as the proof-of-concept, the Bacillus subtilis DivIVA, a protein that can sense negative membrane curvature, is used for anchoring the pole to overcome rapid protein diffusion in the cytoplasm. Thereafter, asymmetric localization of the reporter protein is clearly observed, which reveals that the effect is inherited by only one of the two daughters upon cell division. By using MS2 stem loops to visualize asymmetric transcriptional activity in live cells, we realize functional differentiation by replacing the fluorescent protein with β-lactamase, which also provides ampicillin resistance to only one of the two daughters under the threat of antibiotics. This is, to the best of our knowledge, the first reconstruction of asymmetric cell division and a milestone in the field of synthetic biology. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72213 |
DOI: | 10.6342/NTU201803180 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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