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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃筱鈞(Hsiao-Chun Huang) | |
| dc.contributor.author | Jui-Chung Hong | en |
| dc.contributor.author | 洪瑞鍾 | zh_TW |
| dc.date.accessioned | 2022-11-23T09:12:15Z | - |
| dc.date.available | 2021-08-13 | |
| dc.date.available | 2022-11-23T09:12:15Z | - |
| dc.date.copyright | 2021-08-13 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-10 | |
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Levchenko, I., Seidel, M., Sauer, R. T., Baker, T. A. (2000). A specificity-enhancing factor for the ClpXP degradation machine. SCIENCE, 289(5488), 2354-2356. Lin, D. W., Liu, Y., Lee, Y. Q., Yang, P. J., Ho, C. T., Hong, J. C., . . . Huang, H. C. (2021). Construction of intracellular asymmetry and asymmetric division in Escherichia coli. Nat Commun, 12(1), 888. MacRae, J. D., Smit, J. (1991). Characterization of Caulobacters Isolated from Wastewater Treatment Systems. American Society for Microbiology, 57, 751-758. Martin, S. G., Berthelot-Grosjean, M. (2009). Polar gradients of the DYRK-family kinase Pom1 couple cell length with the cell cycle. Nature, 459(7248), 852-856. McGinness, K. E., Baker, T. A., Sauer, R. T. (2006). Engineering controllable protein degradation. Mol Cell, 22(5), 701-707. Moseley, J. B., Mayeux, A., Paoletti, A., Nurse, P. (2009). A spatial gradient coordinates cell size and mitotic entry in fission yeast. Nature, 459(7248), 857-860. Radhakrishnan, S. K., Thanbichler, M., Viollier, P. H. (2008). The dynamic interplay between a cell fate determinant and a lysozyme homolog drives the asymmetric division cycle of Caulobacter crescentus. Genes Dev, 22(2), 212-225. Ramamurthi, K. S., Losick, R. (2009). Negative membrane curvature as a cue for subcellular localization of a bacterial protein. Proceedings of the National Academy of Sciences of the United States of America, 106(32), 13541-13545. Shetty, R. P., Endy, D., Knight, T. F., Jr. (2008). Engineering BioBrick vectors from BioBrick parts. J Biol Eng, 2, 5. Strutt, D. I. (2002). The asymmetric subcellular localisation of components of the planar polarity pathway. Seminars in Cell Developmental Biology, 13(3), 225-231. Sundararajan, K., Goley, E. D. (2017). Cytoskeletal Proteins in Caulobacter crescentus: Spatial Orchestrators of Cell Cycle Progression, Development, and Cell Shape. Subcell Biochem, 84, 103-137. van Dop, M., Fiedler, M., Mutte, S., de Keijzer, J., Olijslager, L., Albrecht, C., . . . Weijers, D. (2020). DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes. Cell, 180(3), 427-439 e412. Wang, H. R., Zhang, Y., Ozdamar, B., Ogunjimi, A. A., Alexandrova, E., Thomsen, G. H., Wrana, J. L. (2003). Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. SCIENCE, 302(5651), 1775-1779. Waugh, D. S. (2011). An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif, 80(2), 283-293. Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grunewald, S., Scheek, S., . . . Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nat Methods, 3(12), 985-993. Yan, D., Guo, L., Wang, Y. (2006). Requirement of dendritic Akt degradation by the ubiquitin-proteasome system for neuronal polarity. J Cell Biol, 174(3), 415-424. Yoshida, S., van der Schuren, A., van Dop, M., van Galen, L., Saiga, S., Adibi, M., . . . Weijers, D. (2019). A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis. Nat Plants, 5(2), 160-166. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79811 | - |
| dc.description.abstract | "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。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-23T09:12:15Z (GMT). No. of bitstreams: 1 U0001-0708202102362600.pdf: 3807907 bytes, checksum: 02bc5e6caed75d4fb0562a767d80b905 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 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 | |
| dc.language.iso | en | |
| dc.subject | 蛋白質局部降解 | zh_TW |
| dc.subject | 新月柄桿菌 | zh_TW |
| dc.subject | 大腸桿菌 | zh_TW |
| dc.subject | 極化蛋白 | zh_TW |
| dc.subject | 菸草蝕刻病毒蛋白酶 | zh_TW |
| dc.subject | 降解決定子 | zh_TW |
| dc.subject | 細胞不對稱分裂 | zh_TW |
| dc.subject | Caulobacter crescentus | en |
| dc.subject | Proteolysis | en |
| dc.subject | Escherichia coli | en |
| dc.subject | Degron | en |
| dc.subject | Asymmetric cell division | en |
| dc.subject | Tobacco etch virus protease | en |
| dc.title | 在大腸桿菌中利用調控空間的蛋白質降解建立細胞中的不對稱性 | zh_TW |
| dc.title | Construction of intracellular asymmetry using spatially regulated proteolysis in Escherichia coli | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳亘承(Hsin-Tsai Liu),史有伶(Chih-Yang Tseng) | |
| dc.subject.keyword | 細胞不對稱分裂,新月柄桿菌,大腸桿菌,極化蛋白,菸草蝕刻病毒蛋白酶,降解決定子,蛋白質局部降解, | zh_TW |
| dc.subject.keyword | Asymmetric cell division,Escherichia coli,Caulobacter crescentus,Tobacco etch virus protease,Degron,Proteolysis, | en |
| dc.relation.page | 75 | |
| dc.identifier.doi | 10.6342/NTU202102169 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-08-12 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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