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標題: | AraC正迴饋線路促使細胞間變異性放大提供細胞族群之生存優勢 AraC Positive Feedback Loop amplifies Cell-Cell Variation to provide survival advantage to a clonal population |
作者: | Ying-Yu Jiang 江瑩育 |
指導教授: | 黃筱鈞(Huang, Hsiao-Chun) |
關鍵字: | 正回饋基因調控迴路,噪音放大,細胞間變異,阿糖胞?,Gillespie 演算法,抗藥性變異, cell-cell variation,positive feedback loop,noise amplification,antibiotic gene, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 正回饋基因調控迴路與未調控基因在基因轉錄網絡中相較之下,
正回饋基因調控迴路具有噪音放大的特性,此特性會造成相同基因組成的細胞在基因表達量上產生更大的變異。而細胞間變異為此細胞族群在變化萬千的環境中提供更具優勢的生存機會。然而,正回饋基因調控迴路與未調控基因相比之下,同時也具有基因高表達量的特性,因此,為避免基因高表達量造成生存優勢上的主因,我們試圖調控正回饋基因調控迴路上之啟動子強弱,藉此降低正回饋基因表達量與未調控基因相同。在我們的研究項目當中,我們選擇在大腸桿菌中建構阿糖胞苷正回饋表達系統,首先,我們在阿糖胞苷啟動子上設計一系列與核醣核酸聚合酶較弱親和性之核酸序列,藉此減弱阿糖胞苷正回饋之強度達到降低整體基因表達量的目的,而此研究使用紅色螢光蛋白為同步報導阿糖胞苷表現之螢光蛋白。另外一方面,我們使用Gillespie 演算法模擬啟動子強度及阿糖胞苷的親和性和協同性不同參數預測正回饋基因與未調空基因在平均蛋白表達量的一致,接著,藉由模擬預測結果在大腸桿菌中設計並植入阿糖胞苷正回饋線路的核酸質體,最後由流式細胞儀及影像分析軟體透過紅色螢光報導基因分析阿糖胞苷之表達變異性。相信再過不久的將來,我們能夠替換紅色螢光報導基因,研究噪音放大效應對抗藥性變異特性等生存優勢之關係。 Compared to an unregulated gene, auto-regulation or positive-feedback loop (PFL) in a transcriptional network is known to have the characteris¬tic of noise amplification, leading to larger expression variations for cells with identical genetics. A high cell-cell variation potentially offers greater chance of survival for the population in a changing environment. PFL, however, also increases mean expression level, making it difficult to conclude the survival advantage comes from greater variation. To over¬come this, we aim to normalize the expression level of PFL to that of an unregulated gene. We chose AraC expression in E coli as our model system, and designed a series of AraC and RNA polymerase bind¬ing sites in AraC promoter to provide a spectrum of binding affinity and thus expression/feedback strengths. RFP was used as to report expres¬sion level. We also used Gillespie algorithm to predict which combina¬tions of -35 and/or -10 regions (according to the Anderson library) and AraC cooperativity/affinity that would most likely to generate same pro¬tein expression for unregulated gene and PFL. With these predictions, we constructed plasmids, transformed them into E coli, and analyzed RFP strength by flow cytometry, and will further confirm if these data agrees with our computational predictions. Our ultimate goal is to replace RFP with an antibiotic gene (e.g. ampicillin), and test if a larger expression of this resistance gene provides greater survival advantages with a fluctuat¬ing/higher ampicillin concentrations. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55574 |
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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