利用合成系統在大腸桿菌中實現和優化空間的不對稱性

Abstract

不對稱的分裂使細胞得以產生不同功能的子細胞，並以此因應環境的變化，來維持自身的生存以及發育。也因此，不對稱性分裂廣泛存在於各式各樣的生命系統當中，幾乎已經是真核生物的基本特性。不管是在單細胞或是多細胞中，大多數的生物都具有此驚人的特性，將物質在空間上不對稱地分佈並進一步進行不對稱分裂；為了更好地了解這樣的系統，我們選擇了本來就不具有不對稱性分裂的大腸桿菌作為本篇研究的模式生物，利用合成生物學的概念，將不對稱性分裂系統建立在其體內作為平台以進行研究。平台之中包含了來自新月柄桿菌的極區組織蛋白PopZ以及附著端溶菌酶SpmX蛋白，利用PopZ自我聚集在細胞端點的特性作為細胞不對稱性的基礎，配合SpmX的N端（SpmX△C）與PopZ的結合，並進一步將SpmX△C與T7核糖核酸酶融合作為轉接媒介，最後轉錄出下游訊號細胞分裂蛋白DivIVA，而DivIVA也隨即在PopZ端被轉譯成蛋白質，因此當細胞分裂時，兩個子細胞擁有不同的蛋白組合，進而成功實現此平台。而為了優化這個平台，我們也採取了多種方式，諸如調整核糖體結合位點、在蛋白質上接上降解標籤、更換轉接媒介；甚至透過ZipAC將黴漿菌Lon蛋白酶（mfLon protease）建立在細胞中點，以作為下游的拮抗訊號。統合以上所述，此篇研究展示了在大腸桿菌上建立合成性的不對稱分裂平台。

Asymmetric cell division gives rise to two daughter cells with distinct cell fates in response to their environment in order to maintain certain key functions critical for their survival and development. Asymmetric cell division is ubiquitous and arguably one of the most fundamental properties of living systems. From unicellular to multicellular organisms, most organisms have the astonishing capability to prompt the spatial asymmetry of molecules and then implement asymmetric cell division. To better understand this system, we chose a model organism lacking asymmetric division per se, which is the E. coli from prokaryotes, then took a synthetic approach to engineer the asymmetric division platform into E. coli. In this synthetic system, we first utilized PopZ from Caulobacter crescentus as fate-determinant in E. coli, where PopZ self-assembled into a macromolecule to localize at one pole. We then took SpmX from C. crescentus as an adaptor, which the N-terminus (SpmX△C) was capable to be recruited by PopZ. Hereafter, SpmX△C fused with Split T7 RNAPs reconstituted into a functional enzyme, which transcribed downstream gene of interest (reporter) divIVA. DivIVA mRNAs were immediately translated to functional proteins that were retained at the PopZ pole via its negative curvature membrane targeting capacity. Next, we applied multiple strategies to refine our synthetic asymmetric platform, such as tuning ribosome binding site, adding degradation tag and replacing the adaptor. Furthermore, we added mf-Lon protease as antagonizing signal of downstream signal localized at mid-cell via ZipAC’. In sum, we showcased the building of synthetic biological asymmetry from scratch.