在大腸桿菌平台中實現功能分化

Realization of Functional Differentiation in Escherichia coli

何佳澤(Chia-Tse Ho)

指導教授 ： 黃筱鈞

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900988

摘要

大腸桿菌在一般的認知下，不論是形態上或是功能上都被定義成典型的對稱分裂例子。本研究中，希望能夠由合成生物學的角度出發，實現大腸桿菌在形態上或功能上的不對稱分裂。本研究是以PopZ作為主導蛋白，它擁有在細胞的其中一端聚合成大分子的能力，根據此特性，希望可以找到一個能與PopZ交互作用的蛋白，在細胞的端點與PopZ形成複合體，並將這個蛋白作為轉接器，讓轉接器攜帶一與之融合的蛋白至此端點，藉此達到細胞分子的不對稱。
　　在本研究中，是以SpmX作為轉接器，試圖建構出一個以PopZ作為主導蛋白的平台，並將此平台與分割成兩部份的T7 RNA聚合酶作配合，設計出一套基因模組，能夠讓基因在空間上做到不對稱的表達，為印證此系統的可行性，我們在T7下游的報告系統中選用了DivIVA這個蛋白質作為主要的觀測對象。前人的研究指出，DivIVA屬曲率敏感型蛋白質，會在細胞中曲率最大的位置形成複合體，一般而言，DivIVA在桿狀的大腸桿菌中應是對稱表達在兩個極點的。在先前的實驗，我們以綠螢光標記DivIVA，透過PopZ以及SpmX系統的誘導，已成功打破DivIVA的對稱性，看到綠螢光在細胞分裂前的不對稱分佈。
　　上述實驗有了雛型，能夠看到綠螢光在兩個極點的不對稱分佈，但為了實現功能性的不對稱，還需要進一步的優化。因此我們利用了CpdR，另一種會與PopZ形成交互網絡的蛋白質，取代了其中一邊的SpmX，以避免SpmX自我吸引、堆疊的情形，試圖降低RNAP活性被隨機激活的比例。而調整後的結果也顯示，DivIVA在極點的表現量的確達到了優化，背景值明顯降低許多。
　　在功能性表達實驗中，我們選用了盤尼西林酶作為報告者，藉由NSCC/PopZ系統的推動，希望能在細胞單極點做出盤尼西林酶，分裂後僅讓一隻子細胞繼承，隨後再加入安比西林，觀察兩個子細胞的命運是否因此不同，藉此印證大腸桿菌是否達到功能性不對稱分裂。從統計結果發現，具有盤尼西林抗性的細胞能夠存活的時間更長，印證了我們的系統的確做到了某種程度的功能分化。

關鍵字

合成生物學 ； 功能性不對稱分化 ； 極性蛋白 ； 系統優化 ； 細胞死亡

並列摘要

In the previous studies, E. coli is defined as a classic example of symmetric cell division. Based on the principle of synthetic biology. we aim to break this balance and realize the asymmetric cell division in E. coli platform. We use the PopZ protein as the organizer, it has the property to self-organize and fold into the higher-level structure at the cell pole. We plan to find a protein which can be recruit by PopZ as our adaptor and then fuse another target with it. By this way, the target will be brought to the cell pole via PopZ recruitment, thus, the molecular asymmetry is built.
　　In this study, we choose SpmX as our adaptor in the beginning. We cut the RNA polymerase (RNAP) into two subunits and fused them with the SpmX individually. By this way, we hope the activity of RNAP can be re-activated at the pole where the PopZ is localized. Thus, we can realize the transcriptional asymmetry. In this experiment, we choose the DivIVA protein as our reporter, the gene expression of which is driven by RNAP. In the previous study, DivIVA is a curvature sensitive protein, which has the property to accumulate and oligomerize at both endpoints in the rod-shaped bacteria such as E. coli, it should be symmetric distribution. To confirm our assumption is correct, we fuse the DivIVA with the superfolder GFP and try to drive its expression by PopZ/SpmX system. The results indicate that we success to break the symmetric distribution of DivIVA. The transcriptional asymmetry can be built by this system.
　　However, if we want to build the functional asymmetry, this system is still not robust enough. Afterward, SpmX is indicated that it has the property to self-organized. It may cause the activity of RNAP is activated by random. To prevent this situation, we decide to replace one of the SpmX to another adaptor, CpdR. After adjusting, the new NSCC/PopZ system shows the lower noise signal, and the strength of unipolarity has been optimized.
　　To realize the functional differentiation, we use the beta-lactamase (AmpR) fused with DivIVA as our reporter. We hope that we can build the asymmetric gene expression of the reporter by NSCC/PopZ system. We assume that one of the daughter cells can inherit beta-lactamase by cell division, which has a higher survivability in the condition with Ampicillin. After a series of experiment, we find that both of the daughter cells will die after treating with Ampicillin. However, the cell which inherits the beta-lactamase can survive for a longer time. To some degree, this result confirms that we have realized the functional differentiation in the E. coli platform.

並列關鍵字

Synthetic biology ； functional differentiation ； cell polarization ； PopZ ； SpmX ； DivIVA ； asymmetric cell division ； cell death

參考文獻

Reference

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