臺灣博碩士論文加值系統

細胞不對稱分裂對於生物的生長是一個很重要的過程，影響著原核和真核生物子細胞的分化和功能。自然界中生物會透過基因線路的表現或蛋白質層次進行調控。其中，蛋白質層次的調控比起基因線路的表現所需的作用時間更短，例如蛋白質局部降解也是調控極化訊號的重要特性之一。先前我們已經利用新月柄桿菌(Caulobacter crescentus)的極化蛋白，PopZ和SpmXΔC，建立出細胞中的不對稱性，PopZ作為極化聚集的中心，能夠聚合成大分子集中在細胞的其中一端，且會吸引其他蛋白質聚集，適合作為支架蛋白。SpmXΔC作為轉接子蛋白能夠被PopZ吸引形成極化複合蛋白。透過PopZ/ SpmXΔC極化系統，使得切半的T7噬菌體RNA聚合酶重新活化，能從PopZ端開始轉譯出下游報導系統的蛋白，最後在大腸桿菌(Escherichia coli)中建立了不對稱性和細胞分裂後的功能性分化。本研究中，我們想以PopZ/SpmXΔC為平台，建立蛋白質局部降解的不對稱性分裂。利用煙草蝕刻病毒蛋白酶(Tobacco etch virus protease,TEVP)進行蛋白質水解的功能。將分割成兩半的TEV蛋白酶與SpmXΔC融合後，透過PopZ和SpmXΔC的交互作用被攜帶到細胞的PopZ端，重新組裝後活化，TEV蛋白酶能夠辨識特定的胺基酸序列並進行切除，透過此基因模組，使得切半的TEV 蛋白酶被限制在細胞其中一個極端，重新活化後進而剪切特定的胺基酸序列。我們在下游的報導系統選用具有正交性的DivIVA蛋白質，由於DivIVA蛋白質的特性為負曲率敏感，會聚集在細胞的兩個極端，所以利用DivIVA蛋白質限制與之融合的綠色螢光蛋白(GFP)擴散速率。接著我們在GFP後面加上一個降解決定子(degron)，並在他們之間插入TEV 蛋白酶辨識的胺基酸序列。因此，當切半TEV 蛋白酶在細胞中PopZ聚集處重新組裝後，辨識到特定的胺基酸序列並將degron切除，報導系統中的GFP才會穩定表現，而細胞中另一端維持蛋白質不穩定的狀態，接著被降解。通過PopZ/ SpmXΔC)的基因模組，觀察報導系統中螢光分布的情形，我們成功實現了在大腸桿菌中建立細胞內蛋白質局部降解的不對稱性。

Asymmetric cell division is an important process for development. In nature, many cellular functions can be regulated at the level of gene regulation or post-translationally. Protein-level regulations could offer a potential advantage in the speed of operation compared to gene regulation circuits, and are often found in cel-lular processes that required rapid dynamics. We have previously described polarity module based on two central polarity proteins in Caulobacter crescentus, PopZ and SpmX, and used the module to reconstitute split T7 RNA polymerases and activate gene expression asymmetrically, leading to intracellular asymmetry and asymmetric division in Escherichia coli. In this study, we devised a series of experiments to sys-tematically investigate whether localized proteolysis can also be employed to con-struct intracellular asymmetry in Escherichia coli. The same PopZ-based polarity system was adopted, and the split Tobacco etch virus proteases (TEVP) was used as the enzyme for proteolysis. A TEVP cleavage site was inserted between a reporter protein and a tested C-terminal degron. When TEVP was reconstituted via the PopZ scaffold, the unstable reporter could be locally stabilized, leading to an exclusive lo-calization at the PopZ pole. We envision that circuits designed in this study would help to expand the synthetic biology repository for the engineering of synthetic mor-phogenesis, particularly for processes that require dynamic control of local protein abundance.

致謝 1
摘要 3
Abstract 5
Contents 7
Figure List 9
Table List 10
Chapter 1. INTRODUCTION 11
1.1 Synthetic biology 11
1.2 The importance of cellular asymmetry 11
1.3 Protein degradation 12
1.4 Caulobacter crescentus 13
1.5 PopZ and SpmX 14
1.6 TEV protease 15
1.7 DivIVA 16
1.8 Degradation tag 17
Chapter 2. METERIAL AND METHODS 19
2.1 Bacteria strain 19
2.2 Medium 19
2.3 Extraction system for plasmid DNA 20
2.4 Gel extraction system for DNA fragment 21
2.5 PCR clean up system 23
2.6 Concentration of antibiotic 24
2.7 Polymerase chain reaction (PCR) 24
2.8 Circular polymerase extension cloning (CPEC) 25
2.9 Plasmid DNA digested by enzyme (Biobrick assemble) 26
2.10 Ligation 27
2.11 Plasmid transformation 28
2.12 Statistical analysis 28
2.13 Primer design 29
2.14 Fluorescence microscopy 32
Chapter 3. RESULTS 34
3.1 Construct the PopZ-expressing circuit 34
3.2 Validation of SpmXΔC as an adaptor for PopZ 35
3.3 Utilizing DivIVA to target cell poles 38
3.4 Achieve intracellular asymmetry using spatially regulated proteolysis via Tobacco etch virus proteases 38
3.5 A strong degradation tag to eliminate the reporter protein 40
3.6 Validation of whether the cutting site can be recognized by TEV protease 41
3.7 The reconstitution of split TEV protease via the PopZ/ SpmXΔC system 43
3.8 Construction of localized proteolysis with validated modules 45
3.9 The procedures of time-lapse microscopy 46
Chapter 4. CONCLUSION AND FUTURE WORK 49
Figures 50
Reference 73

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