利用調控時間與空間的蛋白質平台在大腸桿菌細胞中建立人工的不對稱性

Abstract

為了在大腸桿菌(Escherichia coli)細胞中建立細胞的不對稱性，我們利用星月鼠桿菌(Caulobacter crescentus)中的PopZ與SpmX蛋白設計基因線路，其中，PopZ能夠聚合成為大分子並即化在細胞的其中一端，同時能夠招募SpmX形成極化的複合蛋白。根據這項特性，本研究將SpmX作為轉接器，能透過PopZ與SpmX的交互作用，攜帶任一與之融合的蛋白至細胞具有PopZ聚集之處。配合隨機模型的幫助，我們了解到類核區排擠效應(nucleoid occlusion)有相當程度的影響了PopZ的極化過程，並引導我們如何調控PopZ的極化。在先前的結果中發現，PopZ即便是在E. coli中也能形成單極化的聚集，且能招募SpmX，這項結果使我們確信PopZ/SpmX平台的設計方向理應能夠操控任何蛋白的分布，但為了證明並演示PopZ/SpmX平台的應用性，我們根據雙分子螢光互補試驗(Bimolecular Fluorescence Complementation assay, BIFC)讓分割成兩部分的eYFP能藉由我們的平台而能重組並能激發其螢光，在顯微鏡觀察的結果能發現eYFP的螢光甚至與PopZ的位置重疊。我們接著使用類似的策略，將PopZ/SpmX平台配合分割成兩部分的T7 RNA聚合酶或TEV蛋白酶，設計出兩種基因模組，前者能誘導基因在細胞空間中不對稱表達，後者則是被限制在細胞極端才能剪切特定胺基酸序列。然而在目前的結果中，仍無法在蛋白質的層級觀察到基因於空間中不對稱的表達，我們尚須需要找尋合適的報導系統，例如直接標定T7 RNA聚合酶發生轉錄之位置。最後，我們嘗試模仿星月鼠桿菌不對稱分裂的過程，在將要分裂的細胞兩端堆積不同的蛋白質組成，而我們則是利用抑制PopZ的表達，以使子細胞與母細胞之間產生形態上的不同，這項結果成功地在顯微鏡下連續紀錄中發現。藉由學習細胞中已存在的機制，配合合成生物學的方法與概念，證實了PopZ/SpmX平台能幫助我們操縱蛋白在細胞中不均勻的分布，進而產生細胞的不對稱性，且由於恢復split eYFP功能的完成，我們更是期待能利用PopZ/SpmX平台在細胞空間中產生不對稱的功能，甚至令分裂後的子細胞展現出與母細胞完全不同的功能性。

To implement cellular asymmetry in E. coli, we designed a spatiotemporal regulatory circuit based on a pair of interacting proteins, PopZ and SpmX, cloned from Caulobacter crescentus. Here, PopZ serves as the actuator capable of self-assembling at one pole of a cell, and SpmX is responsible for recruiting protein of interesting (POI) to the pole with the PopZ foci. With a stochastic computational model, we showed that nucleoid occlusion is critical for the polarity of PopZ, and provided insights into how to control the polarity. Bimolecular Fluorescence Complementation assay (BIFC) was used to demonstrate the ability of our PopZ/SpmX platform to recruit and assemble split eYFP at the pole with unipolarized PopZ. We then tested if the platform can be used to recruit and assemble other split proteins to execute asymmetric functions. Split T7 RNA polymerase and split TEV protease were chosen to implement asymmetric gene expression and peptide cleavage, respectively. In the case of split T7 RNA polymerase, although we have not yet observed asymmetric gene expression at the protein level, it is possible that the asymmetry can be reported with other assays, for instance by monitoring transcription. Finally, we tried to realize asymmetric cell division, i.e. difference in cell fates/morphologies between two daughter cells, by differentially depositing proteins at two poles of a predivisional cell. We showed that after a PopZ focus has formed at a single pole, with a regulatory circuit we can block PopZ expression to generate two distinct daughters, i.e. the PopZ focus was inherited by only one of the two daughter cells. In sum, with lessons from nature and paradigms of synthetic biology, we have characterized a platform to promote cellular asymmetry by manipulating the spatial distribution of proteins. With the success in functionalizing split eYFP, we are hopeful that the platform can be used to promote asymmetric functions and ultimately, functional difference between two dividing daughters.