奠基於PopZ的細胞極化模組──空間上基因表現調控的全新方法

Abstract

合成生物學(synthetic biology)領域中有許多能控制蛋白質表達位置的工具。然而，這些工具多是將蛋白產物送往指定的目的，如特定胞器、細胞膜或者細胞外，而非一個空間上特定的區域。本研究所介紹的奠基於PopZ之細胞極化模組(PopZ-based cellular polarity module)，能夠將目標蛋白送往細胞的一端或兩極處，進而使得在空間上調控基因表現達到史無前例的嶄新層級。 我們在大腸桿菌(Escherichia coli)中，表現新月柄桿菌(Caulobacter crescentus)的蛋白，藉以測試它們在正交環境(orthogonal context)中的適用性。其中，以PopZ做為細胞極的燈塔，並以SpmX的muramidase domain作為轉接頭，將其他作用蛋白帶往受PopZ標定的細胞極。我們首先分析這些蛋白的分布狀態與表現量、寡聚化效率(oligomerization efficiency)、SpmX完整性與稀釋速率等四項參數間的關係，並找出達到雙極化、單極化與四散分布的條件。此外，我們也以流式細胞儀與縮時顯微攝影，證明在特定條件下，PopZ在兩個子細胞間呈現不對稱分布。 接下來，我們探討如何實現本模組的三大特色：支架、極化與不對稱分裂。劉揚已於2015年以BiFC證實PopZ寡聚體能夠作為支架蛋白，讓標的蛋白之間的作用機會在局部提升；而藉由微米級細胞光電單元(micron-level cellular photovoltaic unit, MCPU)的企劃，我們討論此系統將胞內分子極化的應用性。最後，對於實現人工不對稱分裂，本文提出可能的作法，並佐以初步實驗與動態模擬結果，以期能利用T7 ploymerase與mfLon protease完成這個目標。 本研究的成果，提供合成生物學社群平台，使得更加複雜的系統成為可能。另一方面，不對稱細胞分裂這個充滿待解謎團的現象，亦隨著本研究結果的出爐，顯得更能夠捉摸。

In synthetic biology, there are many techniques to control where protein products are expressed. However, most of these tools only specify a certain target (eg. organelles, membrane or outside of the cell), instead of a spatially defined region. Our study, PopZ-based cellular polarity module, is able to localize cellular components to one or both cell poles, and provide a way to achieve an unprecedented level for spatial regulation of gene expression. In this system, Caulobacter crescentus proteins are expressed in Escherichia coli to test their applicability in an orthogonal context. PopZ is adopted as the cell pole identifier, while SpmX’s muramidase domain is used as an adapter to bring effector proteins toward the PopZ-tagged pole. We first characterized the distribution of heterologously expressed molecules against four parameters: expression level, oligomerization efficiency, SpmX intactness and dilution rate, and found conditions where PopZ became either bipolar, unipolar or diffused. Furthermore, by using flow cytometry and time-lapse imaging, we demonstrated that under certain conditions, the introduced proteins could be asymmetrically partitioned to the daughter cells. Next, we showcased three main features of the module: scaffold, polarization and asymmetric cell division. In 2015, Liu showed that PopZ aggregation can act as a scaffold to locally increase the interaction of target proteins using a split yellow fluorescent protein. We further demonstrated that our system can polarize cellular components of interest, using bacteriorhodopsin-based “micron battery” as an example. Moreover, T7 polymerase and mfLon protease were utilized to show that the system can also accomplish asymmetric cell division. The results of this study present a promising platform on which the synthetic biology community can build more complicated systems. On the other hand, they also provide useful insights to asymmetric cell division, unveiling this perplexing phenomenon yet a step further.