合成性細胞不對稱系統的改造與優化

Abstract

細胞的各種行為表現一直以來都為科學家汲汲營營地探求。在多數研究者著墨於調控基因產物的表達以了解細胞行為的改變時，合成生物學家則試圖利用該領域備有之眾多工具來從無到有重組出該現象。在萬端細胞行為中最有趣的莫過於不對稱細胞分裂，於合成生物學的終極目標——人工合成生物——更是不可或缺的一環。為此，我們在大腸桿菌（Escherichia coli）中構築了具有極區組織能力的平臺。 我們引進新月柄桿菌（Caulobacter crecentus）中聚集於極區的兩種蛋白質，分別是極區組織蛋白PopZ以及附著端溶菌酶同源蛋白SpmX。由於極區組織蛋白在大腸桿菌中呈穩定單極化分佈，我們將其作為雛形平臺的支架；至於附著端溶菌酶同源蛋白，我們則去除其穿膜能力、存乎其溶菌酶區域（muramidase domain），作為其他蛋白和極區組織蛋白作用的轉接媒介。經過初步試驗，我們得知將兩者結合確實能將後者所搭載的其他因子拉近距離，從而在極區組織蛋白周遭結合組裝。我們將即進一步擴展物理性的蛋白質團不對稱至實質功能的不對稱。 此研究中引用了數種外源蛋白和合成工具來測試和優化平臺雛形。首先，我們為了測試T7核糖核酸聚合酶（T7 RNA polymerase）與平臺的相容性而更換了下游表達蛋白，又為提升下游蛋白的影響而引入黴漿菌Lon蛋白酶（mfLon protease）。此外，我們還使用了MS2噬菌體蛋白（MS2 coat protein）殼來觀測轉錄活動。於此同時，我們對附著端溶菌酶同源蛋白的溶菌酶區域做了截斷蛋白（protein truncation）試驗，試圖去蕪存菁純化其和極區組織蛋白作用的功能。我們亦嘗試利用合成拉鍊工具（SYNZIP）組來調整極區組織蛋白的結合能力。 綜合以上改動和測試，我們對於此平臺的性能優劣和潛力有了相當程度的理解。據此，我們將以細胞級化平臺為基，繼以合成生物學的方法發展之，希冀能藉此合成人工的不對稱細胞分裂。

Scientists have been endeavoring to explain how various cell behaviors are brought about. While some researchers comprehend life phenomena by regulating gene products, synthetic biologists have dedicated to recreating them with their synthetic tool miscellany. Among all, the realization of asymmetric cell division will lead to the most progress in achieving the final goal, synthesis of life. In the light, our research group constructed a polar-organized platform in Escherichia coli, making use of biological parts from other organisms. The prototype of the platform is organized with two Caulobacter crecentus proteins, PopZ the scaffold and SpmX the source of the adapter. PopZ, displaying a stable unipolarity in E.coli, is capable of recruiting the muramidase domain of SpmX (SpmX450) hence enforcing proximity to the factors it carries. Our subsequent objective is to extend the physical PopZ-SpmX asymmetry into functional asymmetry in the model system. For this purpose, we employed various exogenous components and synthetic elements into our platform in this study. We first tested the adaptability of split T7 RNA polymerase system with different downstream reporters and introduced mfLon protease to elevate their influences. We further utilize the MS2 assay to identify where transcription brings about. On the other hand, truncations were performed on SpmX450 to refine its role as an adapter. Attempts were also made to regulate PopZ’s binding ability with the Synzip toolbox. With all the efforts, the potentials and the limits of our platform are both revealed. The comprehension will yet give rise to new applicable ideas leading to our goal, i.e. establishment of an asymmetric division platform.