在大腸桿菌中建立光調控局部蛋白質調控系統

Abstract

細胞的不對稱分裂與分化是發育生物學中一個長久的關注議題。細胞透過極化並進行不對稱分裂生成兩個具有不同發展宿命的子細胞，進而達成細胞種類的多樣化。此外，細胞也透過極化執行各種功能，比如移動與出芽生殖。 細胞極性的產生可受外部因子調控亦可由內部因素引發並透過不同的途徑產生下游物質的不對稱分佈，比如透過極性分布的RhoA GTPase調控細胞骨架進而控制細胞移動，又或者透過Bicoid mRNA透過極性分布產生Bicoid morphongen的濃度梯度決定果蠅胚胎各部位分化的位置等。 在先前的研究中，我們實驗室已經透過在大腸桿菌中異源表達新月柄桿菌聚合蛋白PopZ證實單極的正回饋可以作為一個最基本的功能單元成功在對稱的細胞內建立極性並進行不對稱分裂。 本研究則利用合成生物學試圖於大腸桿菌細胞內透過光遺傳學方法建立細胞極性並驗證交互抑制作用是否也是引發並維持細胞不對稱性所需之最簡化系統。 首先本研究透過外源表達粉紅麵包黴藍光聚合蛋白（Vivid）的靜電荷修飾版本 Magnet作為光誘導聚合因子，並將pMag/nMag雙體中的nMag與枯草桿菌DivIVA蛋白融合，利用DvIVA便是細胞膜負曲率之能力將其固著於細胞兩端。我們首先透過汞燈照射，證實細胞兩端之nMag可以在照光啟動後透過結合將原先分散於細胞中的pMag分布，將之聚集至細胞兩端。接著我們透過458nm氬氣雷射區域性照射，進一步證實Magnet可透過區域性光照在細菌尺度下達成不對稱活化。 之後，本研究透過將split T7 RNA合成酶以及split TEV蛋白酶的N端與C端片段分別與nMag與pMag結合，透過區域性合成與區域性降解兩種途徑試圖在大腸桿菌細胞內產生不對稱的蛋白分布。

Asymmetric division and differentiation of cell is a long standing concern in development biology. Cell divides into two daughter cells that have distinct fate and functions through polarization and asymmetric cell division to increase the cellular diversity. Also, cells use polarization to control many cellular process as well, such as cell migration and budding reproduction. The polarization of cell could be activated through outer stimulation or internal regulation, and generates the asymmetry distribution of downstream substance. For example, the local degradation of RhoA GTPase can regulate the formation of actin fibers and further control cell migration, and the asymmetric distribution of Bicoid mRNA causes the concentration gradient of the Bicoid morphogen that decided the differentiation position of each body part of a fruit fly. In the previous research, our lab has proved that a single positive pole could be the minimal functional unit to establish cellular polarity through heteroexpression of Caulobacter crescentus self-assembly protein PopZ in E. coli, and made the symmetry E. coli cell become functional symmetric division. In this study, we introduced the engineered charged variant of Neurospora crassa Vivid (VVD) blue light sensor, magnet (Mag), into E. coli as a light triger, and fused the nMag in the nMag/pMag light mediated heterodimer with the pole targeting protein DivIVA from Bacillus subtilis to anchor it to the cell pole. We first irradiated the system by short arc lamp to prove the DivIVA fused nMag can recruit the diffused pMag to the cell pole after light activation. Then the system was tested using a 458nm argon laser to achieve local irradiation and prove the Magnet system can be asymmetric activation under bacterial scale. After the local recruitment was confirmed, we fused the Magnet system with both split T7 RNA polymerase and split TEV protease to construct a local protein synthesis and a local proteolysis system for generating asymmetric protein distribution in E. coli.