在大腸桿菌中構建基於細胞極性蛋白質支架的合成生物學平臺

在漫長的生命進化歷史中，由單細胞生命向多細胞生命進化是一段具有里程碑意義的歷程。毫無疑問，這個過程裡最關鍵的要素即為細胞不對稱性和細胞分化行為的出現。細胞的不對稱性賦予了生命在單細胞水準上將生理功能區域化的能力，並且促使細胞發展出分化行為。時至今日，當我們嘗試揭示上述自然哲學真理的本質，適逢合成生物學迅猛發展。合成生物學是一門日益精密的新興學科，致力於通過設計和構建人工基因線路來研究生命科學。一方面，合成生物學嘗試利用合成的基因元件、線路、裝置和系統以研究生命的自然本質。另一方面，研究者們亦在求索的過程中將天然的生物學系統拆分為可替代的標準化基因元件，且利用它們構建出進化所不曾創造生物學系統和生物學功能。從合成生物學的角度出發，在相對簡單的對稱性細胞中構建人工不對稱裝置，對於未來從頭建立人工多細胞生命、探討細胞分化的分子機制起源以及進一步細化工程細胞的生理功能，都有著重要意義。在我們的研究裡，我們在最簡單、應用最廣泛的基因工程學平臺大腸桿菌（Escherichia coli）中，設計並建立了具有不對稱分佈性質的蛋白質支架工具。同時，揭示了這個支架系統的魯棒性和實用性。我們從新月柄桿菌（Caulobacter crescentus）中分離並標準化了自組織蛋白PopZ和膜支架蛋白SpmX的基因，通過實驗證明它們之間存在直接相互作用，還發現了這兩種蛋白在不同基因表達比例下特殊的極性分佈規律。在這些發現的基礎上，我們提出“粘性假說”以闡釋這些獨特現象背後的機制，並由此設計並建立了可誘導的單極-雙極分佈轉換蛋白質支架。為了體現這一支架工具的可用性，我們通過雙分子螢光互補實驗（BiFC）實驗證明PopZ/SpmX支架系統可以有效調控第三方蛋白質的活性。還發現與PopZ的聚合能力可以顯著影響與之融合的抑制子蛋白CI功能域之活性。最後，我們證明了SpmX蛋白質的N末端結構域作為一種蛋白質支架適配子（adapter），具有良好的通用性。我們利用這一支架工具，成功在大腸桿菌（E.coli）細胞基礎上構建了一種微米級細胞光電單元（MCPU）。

關鍵字

合成生物學；細胞極化；蛋白質支架； PopZ ； SpmX ；細菌視紫紅質

並列摘要

In the long history of biological evolution, the evolution of unicellular to multicellular is a milestone event. Undoubtedly, the cornerstone of this event is the emergence of cell asymmetry and cell differentiation. The asymmetry of cells gives cell capacity to divide functional areas at the single cell level, even more, to promote the development process of differentiation behavior. Just today, as we trying to reveal the truths of natural philosophy include above themes, synthetic biology is developing rapidly. Synthetic biology is an increasingly sophisticated emerging discipline that dedicates to study life science by constructing and designing artificial genes circuits. On the one hand, synthetic biologists trying to research the natural essence of life by utilizing synthetic parts, circuits, devices and systems. On the other hand, we also research and split natural gene systems to some replaceable standardized DNA parts, and then, use these parts to design and build unnatural life system with desired functions. In the vision of synthetic biology, building asymmetric device in simple symmetric cell has great significance for construction of multi-cellular system from scratch, researching basic mechanisms of cell differentiation and refinement of artificial function in prokaryotes. In our research, we designed and built asymmetric protein scaffold in the most popular and the simplest single-cell gene engineering platform, E. coli. Also, we verify the practicality and robustness of this scaffold system. We first standardized the self-organizing scaffold proteins PopZ and SpmX from Caulobacter crescentus. Then we prove the existence of the direct interaction between them and test the polarized regularity in different expression proportion of these two genes. Based on our research results, we propose a complete theoretical hypothesis ‘Stickiness hypothesis’ to reveal the rule of the interaction between the two proteins. Based on hypothesis, we make a inducible unipolar-bipolar switch (IUBS) scaffold in E. coli. To prove the availability of this scaffold system, we confirmed that the PopZ can regulate the activity of split EYFP fused with SpmX∆C adapter by a Bimolecular Fluorescence Complementation (BiFC) experiment. Even more, the fusion protein includes N terminus of cI repressor and PopZ can change the expression behavior of the downstream gene circuit. As a scaffold molecule, PopZ can affect the activity of transcription factor. Finally, we prove the universality of SpmX∆C as an adapter then make a Micron Cell-based Photovoltaic Unit (MCPU) to show the powerful feature and versatility of this asymmetry platform.

並列關鍵字

Synthetic Biology ； Cell Polarization ； Protein Scaffold ； PopZ ； SpmX ； Bacteriorhodopsin

參考文獻

1. Purnick, P.E. and R. Weiss, The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol, 2009. 10(6): p. 410-22.

2. Benner, S.A. and A.M. Sismour, Synthetic biology. Nat Rev Genet, 2005. 6(7): p. 533-43.

3. Bornens, M., Organelle positioning and cell polarity. Nat Rev Mol Cell Biol, 2008. 9(11): p. 874-86.

4. Drubin, D.G. and W.J. Nelson, Origins of cell polarity. Cell, 1996. 84(3): p. 335-44.

5. Macara, I.G. and S. Mili, Polarity and differential inheritance--universal attributes of life? Cell, 2008. 135(5): p. 801-12.

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在大腸桿菌中構建基於細胞極性蛋白質支架的合成生物學平臺

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