- 學位論文
在大腸桿菌中建構細胞內的不對稱性和功能性的分化
Construction of intracellular asymmetry and functional differentiation in Escherichia coli
摘要
細胞的分裂與分化不論是在原核生物或是真核生物中都是非常基礎的一個過程。細胞分裂的不對稱性往往是影響子細胞命運和多樣性非常重要的因素。我們利用合成生物學通過人造生物零件重新建構仿生系統的方法來重現細胞中不對稱性的現象。在被作為探討細胞不對稱分裂的模式生物新月柄桿菌中，我們了解到蛋白的局部極化性是影響細胞命運不對稱性的決定因素。為了探討細胞不對稱性分裂的機制以及建構最小基因組來實現細胞不對稱性分裂，我們設計包含了新月柄桿菌蛋白的基因線路，並使用被認定為是對稱性分裂的大腸桿菌作為表現的平台。我們在大腸桿菌中表現極區聚集蛋白(popZ)來做為在大腸桿菌細胞端點穩定聚集的支架蛋白。透過popZ和被其吸引的轉接子蛋白(SpmXΔC)的交互作用以及切半T7噬菌體RNA聚合酶的重新活化的現象，我們使蛋白質從popZ端點開始轉譯，創造了細胞內的蛋白濃度梯度且建立了popZ/SpmXΔC的系統平台。接著透過一個具有正交向的聚合蛋白(DivIVA)以及抗生素抗性基因(AmpC)，我們限制了轉譯出來蛋白的擴散速率，並在細胞分裂之後，提供抗藥性給繼承了popZ蛋白的子細胞。根據報導蛋白(reporter protein)的螢光強度與分布位置，我們發現到蛋白質的極性和不對稱性確實發生在大腸桿菌中。在氨苄青黴素(ampicillin)抗生素的作用之下，子細胞出現了死亡時間的延後與不一致。通過popZ / SpmXΔC系統的平台，我們成功地實現了蛋白質細胞內的不對稱性和細胞分裂後的功能性分化。另一方面，我們希望通過另一種方法，利用煙草蝕刻病毒蛋白酶（Tobacco etch virus protease）的特性來實現在大腸桿菌中蛋白質細胞內不對稱。
並列摘要
Cell division and differentiation are among the most fundamental processes in prokaryotic and eukaryotic cells. Asymmetric cell division is the important process that leads to alternative cell fates and cellular diversity. We applied the strategy of synthetic biology, which is used to create biomimetic systems through artificial biological and chemical building blocks, to bottom-up reconstitute asymmetric phenotypes. In Caulobacter crescentus, a model organism of asymmetric division and cell differentiation, we have learned that polar localization of protein is responsible for the asymmetric segregation of cell fate determinants. To investigate the mechanism of asymmetric cell division and establish the minimal machineries to achieve that, we designed the genetic circuit containing proteins from Caulobacter crescentus, and used the model organism Escherichia coli, which the division process is well-known to be symmetric, as the platform. We expressed a polarity polar organizing protein Z (popZ) as a robust and stable scaffold protein localized in one of E. coli cell poles. Via the polar recruitment of adaptor protein, SpmXΔC, and the polar reconstitution of split T7 bacteria phage RNA polymerase, we asymmetrically expressed proteins from the pole of popZ as our primary platform of popZ/SpmXΔC system. With an orthogonal oligomerization protein, DivIVA, and an antibiotic resistance gene, AmpC, we restricted the diffusion of the translated protein and equipped one of daughter cells that inherited popZ with antibiotic resistance upon cell division. According to the distribution and intensity of fluorescent reporter protein, we concluded that we successfully observed the occurrence of intracellular asymmetry in E. coli. After ampicillin treatment, we also observed a difference in death time between sister cells. In sum, we have successfully achieved intracellular asymmetry and functional differentiation at the first cell division with the platform of popZ/SpmXΔC. In addition, we also explored the possibility of applying the cleaving characteristic of Tobacco etch virus protease (TEV protease) as another strategy to achieve intracellular asymmetry in E. coli.
參考文獻
延伸閱讀
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