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  1. NTU Theses and Dissertations Repository
  2. 生命科學院
  3. 分子與細胞生物學研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87713
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor黃筱鈞zh_TW
dc.contributor.advisorHsiao-Chun Huangen
dc.contributor.author楊承叡zh_TW
dc.contributor.authorCheng-Ruei Yangen
dc.date.accessioned2023-07-11T16:28:17Z-
dc.date.available2024-09-30-
dc.date.copyright2023-07-11-
dc.date.issued2022-
dc.date.submitted2002-01-01-
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Belahbib, H., Renard, E., Santini, S., Jourda, C., Claverie, J.-M., Borchiellini, C., & Le Bivic, A. (2018). New genomic data and analyses challenge the traditional vision of animal epithelium evolution. BMC Genomics, 19(1). doi:10.1186/s12864-018-4715-9
Benner, S. A., & Sismour, A. M. (2005). Synthetic biology. Nature Reviews Genetics, 6(7), 533-543. doi:10.1038/nrg1637
Fahey, B., & Degnan, B. M. (2010). Origin of animal epithelia: insights from the sponge genome. Evolution & Development, 12(6), 601-617. doi:10.1111/j.1525-142x.2010.00445.x
Feng, W., Wu, H., Chan, L.-N., & Zhang, M. (2007). The Par-3 NTD adopts a PB1-like structure required for Par-3 oligomerization and membrane localization. The EMBO Journal, 26(11), 2786-2796. doi:10.1038/sj.emboj.7601702
Heng, B. C., & Fussenegger, M. (2013). Chapter 9 - Design and Application of Synthetic Biology Devices for Therapy. In H. Zhao (Ed.), Synthetic Biology (pp. 159-181). Boston: Academic Press.
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Liu, Z., Yang, Y., Gu, A., Xu, J., Mao, Y., Lu, H., . . . Wen, W. (2020). Par complex cluster formation mediated by phase separation. Nature Communications, 11(1). doi:10.1038/s41467-020-16135-6
Mushnikov, N. V., Fomicheva, A., Gomelsky, M., & Bowman, G. R. (2019). Inducible asymmetric cell division and cell differentiation in a bacterium. Nature Chemical Biology, 15(9), 925-931. doi:10.1038/s41589-019-0340-4
Neumüller, R. A., & Knoblich, J. A. (2009). Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer. Genes & Development, 23(23), 2675-2699. doi:10.1101/gad.1850809
Paintdakhi, A., Parry, B., Campos, M., Irnov, I., Elf, J., Surovtsev, I., & Jacobs-Wagner, C. (2016). Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis. Molecular Microbiology, 99(4), 767-777. doi:10.1111/mmi.13264
Petronczki, M., & Knoblich, J. A. (2001). DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nature Cell Biology, 3(1), 43-49. doi:10.1038/35050550
Purnick, P. E. M., & Weiss, R. (2009). The second wave of synthetic biology: from modules to systems. Nature Reviews Molecular Cell Biology, 10(6), 410-422. doi:10.1038/nrm2698
Renschler, F. A., Bruekner, S. R., Salomon, P. L., Mukherjee, A., Kullmann, L., Schütz-Stoffregen, M. C., . . . Wiesner, S. (2018). Structural basis for the interaction between the cell polarity proteins Par3 and Par6. Sci Signal, 11(517). doi:10.1126/scisignal.aam9899
Renschler, F. A., Bruekner, S. R., Salomon, P. L., Mukherjee, A., Kullmann, L., Schütz-Stoffregen, M. C., . . . Wiesner, S. (2018). Structural basis for the interaction between the cell polarity proteins Par3 and Par6. Science Signaling, 11(517), eaam9899. doi:doi:10.1126/scisignal.aam9899
Saha, S., Weber, C. A., Nousch, M., Adame-Arana, O., Hoege, C., Hein, M. Y., . . . Hyman, A. A. (2016). Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism. Cell, 166(6), 1572-1584.e1516. doi:https://doi.org/10.1016/j.cell.2016.08.006
Salinas-Saavedra, M., Stephenson, T. Q., Dunn, C. W., & Martindale, M. Q. (2015). Par system components are asymmetrically localized in ectodermal epithelia, but not during early development in the sea anemone Nematostella vectensis. EvoDevo, 6(1), 20. doi:10.1186/s13227-015-0014-6
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Wu, H., Feng, W., Chen, J., Chan, L.-N., Huang, S., & Zhang, M. (2007). PDZ Domains of Par-3 as Potential Phosphoinositide Signaling Integrators. Molecular Cell, 28(5), 886-898. doi:https://doi.org/10.1016/j.molcel.2007.10.028
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87713-
dc.description.abstract細胞不對稱分裂顯著影響原核和真核生物的分化和功能,會對後代細胞產生不同的命運,對於生物演化的進程再重要不過。在這些生物體中,一個在演化上相對保守的蛋白質家族,Par 蛋白複合物,參與了細胞質內蛋白質的不對稱分佈和不對稱分裂的控制。而其中PAR-3本身自我聚合的能力以及PAR-3與PAR-6兩個蛋白之間的交互作用是造成不對稱性分布的主要因素,這份研究主要是針對PAR-3與PAR-6進行探討,由於在演化上相當保守,因此我們蒐集了各種不同物種的PAR-3與PAR-6進行本研究的實驗。我們在這份研究中用了三種不同的系統對Par 蛋白複合物進行實驗,分別是在大腸桿菌中表現、在油滴的系統中表現以及蛋白質的實驗分析三種不一樣的實驗系統,透過這三種系統的實驗結果讓我們成功地看見PAR-3與PAR-6兩個蛋白間的交互作用,對於PAR-3本身自我聚合的能力則需要再更進一步地探討,在這三種系統的研究成果中有彼此能夠相互印證的地方也有互相不同的地方。三種系統中也都有各自的問題還必須去解決,因此本研究接下來運行的方向可能會朝向將這三個系統去進行整合,我們必須先解決三的系統中各自的問題然後才能對於三個系統的結果進行比較與統整最終才能更進一步清楚地描繪Par 蛋白複合物整體的運作。zh_TW
dc.description.abstractAsymmetric cell division significantly affects the differentiation and function of prokaryotes and eukaryotes, and produces different fates for daughter cells, which cannot be more important to the process of biological evolution. In these organisms, an evolutionarily relatively conserved protein family, the Par protein complex, is involved in the asymmetric distribution of proteins within the cytoplasm and the control of asymmetric division. The ability of PAR-3 to self-oligomerize and the interaction to PAR-6 are the key factors causing the asymmetric distribution and division. This study mainly focuses on PAR-3 and PAR-6. For discussion, because they are quite conservative in evolution, we collected PAR-3 and PAR-6 from various species for the this study. We used three different systems to perform experiments on Par protein complex, expression in E. coli, droplets, and analysis of proteins. Through these three different systems, the results of the system allow us to successfully find the interaction between the PAR-3 and PAR-6. The ability of PAR-3 to self-aggregate needs to be further explored. The three systems also have their own problems that must be solved, so the direction of this research may be to integrate these three systems. Comparing and integrating the results of the system will eventually lead to a clearer depiction of the overall functioning of the Par protein complex.en
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dc.description.tableofcontents致謝 i
摘要 iii
Abstract iv
Contents vi
Figure List viii
Table List ix
1. Introduction 1
1.1 Synthetic biology 1
1.2 Cell polarity 1
1.3 The important of Asymmetric cell division 2
1.4 PAR (partitioning defective) complex 3
1.5 The structural domains of Par-3 family 4
1.6 Liquid–Liquid phase separation (LLPS) 5
1.7 Asymmetric cell division in non-bilateral animals 6
1.8 Asymmetric cell division in E. coli 7
1.9 Aim 8
2 Materials and methods 10
2.1 Bacteria strains 10
2.2 The culture Medium 10
2.3 Concentration of antibiotic 11
2.4 Bacteria plasmid extraction 11
2.5 Gel extraction for DNA fragment 13
2.6 Primer design 13
2.7 Polymerase chain reaction (PCR) 14
2.8 PCR product clean up 16
2.9 Circular polymerase extension cloning (CPEC) 16
2.10 Restriction enzyme digestion 17
2.11 Agarose electrophoresis 17
2.12 BioBrick Assembly 18
2.13 Ligation 18
2.14 Gibson Assembly 19
2.15 Chemical plasmid transformation 19
2.16 Fluorescent Microscopy 20
2.17 The method of statistical analysis 20
2.18 Primer List for sequencing 20
2.19 Sample lysis 22
2.20 Immunoprecipitation (IP) 22
2.21 Western blotting analysis 23
2.22 In Vitro Transcription/Translation System (IVTT) 31
2.23 Microfluidic chips 31
2.24 Droplet formation 32
3. Result and discussion 33
3.1 The expression in E. coli 33
3.2 Protein gel analysis 41
3.3 The expression in droplet system 46
4. Conclusion and future work 51
5. Figure 53
6. Reference 94
Appendix I. The organizations design of the constructs Par-3N and Par-6β 97
Appendix II. The organizations design of the constructs Par-3 homologous 98
Appendix III. The organizations design of the constructs Par-6 99
Appendix IV. Conserved domains search result of different species Par3 100
Appendix V. Python code 102
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dc.language.isoen-
dc.subject油滴系統zh_TW
dc.subject液-液相分離zh_TW
dc.subject不對稱細胞分裂zh_TW
dc.subject蛋白質聚合zh_TW
dc.subject極化蛋白zh_TW
dc.subject大腸桿菌zh_TW
dc.subjectDroplet formationen
dc.subjectEscherichia colien
dc.subjectAsymmetric cell divisionen
dc.subjectliquid-liquid phase separationen
dc.subjectCell free systemen
dc.subjectPAR complexen
dc.title透過重構Par系統研究不對稱細胞分裂zh_TW
dc.titleInvestigation of asymmetric cell division by reconstituting Par systemsen
dc.typeThesis-
dc.date.schoolyear110-2-
dc.description.degree碩士-
dc.contributor.oralexamcommittee吳亘承zh_TW
dc.contributor.oralexamcommitteeHsiung-Lin Tu;Hsuan-Chen Wuen
dc.subject.keyword不對稱細胞分裂,大腸桿菌,極化蛋白,蛋白質聚合,油滴系統,液-液相分離,zh_TW
dc.subject.keywordAsymmetric cell division,Escherichia coli,PAR complex,Cell free system,Droplet formation,liquid-liquid phase separation,en
dc.relation.page104-
dc.identifier.doi10.6342/NTU202203638-
dc.rights.note同意授權(全球公開)-
dc.date.accepted2022-09-28-
dc.contributor.author-college生命科學院-
dc.contributor.author-dept分子與細胞生物學研究所-
dc.date.embargo-lift2024-09-30-
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