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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃筱鈞(Hsiao-Chun Huang) | |
dc.contributor.author | Chia-Tse Ho | en |
dc.contributor.author | 何佳澤 | zh_TW |
dc.date.accessioned | 2021-06-17T08:17:20Z | - |
dc.date.available | 2024-08-20 | |
dc.date.copyright | 2019-08-20 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-14 | |
dc.identifier.citation | Reference
[1] L. S. Qi et al., 'Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,' Cell, vol. 152, no. 5, pp. 1173-83, Feb 28 2013. [2] A. C. Komor, A. H. Badran, and D. R. Liu, 'CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes,' Cell, vol. 169, no. 3, p. 559, Apr 20 2017. [3] T. S. Gardner, C. R. Cantor, and J. J. Collins, 'Construction of a genetic toggle switch in Escherichia coli,' Nature, vol. 403, no. 6767, pp. 339-42, Jan 20 2000. [4] J. Dworkin, 'Cellular polarity in prokaryotic organisms,' Cold Spring Harb Perspect Biol, vol. 1, no. 6, p. a003368, Dec 2009. [5] R. Li and B. Bowerman, 'Symmetry breaking in biology,' Cold Spring Harb Perspect Biol, vol. 2, no. 3, p. a003475, Mar 2010. [6] M. T. Laub, L. Shapiro, and H. H. McAdams, 'Systems biology of Caulobacter,' Annu Rev Genet, vol. 41, pp. 429-41, 2007. [7] G. R. Bowman et al., 'Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold,' Mol Microbiol, vol. 90, no. 4, pp. 776-95, Nov 2013. [8] G. Laloux and C. Jacobs-Wagner, 'Spatiotemporal control of PopZ localization through cell cycle-coupled multimerization,' J Cell Biol, vol. 201, no. 6, pp. 827-41, Jun 10 2013. [9] S. Saberi and E. Emberly, 'Chromosome driven spatial patterning of proteins in bacteria,' PLoS Comput Biol, vol. 6, no. 11, p. e1000986, Nov 11 2010. [10] A. M. Perez, T. H. Mann, K. Lasker, D. G. Ahrens, M. R. Eckart, and L. Shapiro, 'A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity,' MBio, vol. 8, no. 1, Feb 28 2017. [11] J. A. Holmes, S. E. Follett, H. Wang, C. P. Meadows, K. Varga, and G. R. Bowman, 'Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles,' Proc Natl Acad Sci U S A, vol. 113, no. 44, pp. 12490-12495, Nov 1 2016. [12] J. T. Kadonaga, A. E. Gautier, D. R. Straus, A. D. Charles, M. D. Edge, and J. R. Knowles, 'The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli,' J Biol Chem, vol. 259, no. 4, pp. 2149-54, Feb 25 1984. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74029 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:17:20Z (GMT). No. of bitstreams: 1 ntu-108-R06b43012-1.pdf: 2090861 bytes, checksum: 60c1671cf70c0afe66cac9c48d8de132 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | Table of contents:
中文摘要 6 ABSTRACT 7 CHAPTER 1. INTRODUCTION 9 1.1 SYNTHETIC BIOLOGY 9 1.2 CELL POLARITY AND THE IMPORTANCE OF CELLULAR ASYMMETRY. 10 1.3 ASYMMETRIC CELL DIVISION IN CAULOBACTER CRESCENTUS 10 1.4 SPLIT T7RNAP-SPMXΔC/POPZ SYSTEM 11 1.5 IMPROVEMENT OF OUR ACTUATOR/ADAPTOR PLATFORM SYSTEM 13 CHAPTER 2. MATERIALS AND METHODS 14 2.1 BACTERIAL STRAIN AND CULTURE METHODS 14 2.1.1 Bacteria strain 14 2.1.2 Storage of bacteria 14 2.1.3 Medium 15 2.1.4. Antibiotics usage 16 2.2 DNA METHOD 16 2.2.1 Standard protocol of DNA constructs 16 2.2.2 Primer design 17 2.2.3 PCR methods 17 2.2.4 Electrophoresis and Gel Extraction 18 2.2.5 PCR Clean Up 19 2.2.6 Ligation 21 2.2.7 Transformation 21 2.2.8 Plasmid purification system 22 2.3 CPEC 23 CHAPTER 3. RESULTS AND DISCUSSION 25 3.1 OPTIMIZATION OF THE POPZ DOMINANT SYSTEM 25 3.2 CONSTRUCTION OF FUNCTIONAL DIFFERENTIATION MODULE. 26 3.3 DEVELOPMENT OF MICROSCOPY METHOD 28 3.4 DEGRADATION TAG TEST 30 3.5 REALIZATION OF FUNCTIONAL DIFFERENTIATION 30 3.6 THE TEV PROJECT 32 3.7 DISCUSSION AND FUTURE WORK 33 LIST OF FIGURES 36 FIGURE1. THE LIFE CYCLE OF CAULOBACTER CRESCENTUS. 36 FIGURE 2. AN EXAMPLE OF BIOBRICK STANDARD ASSEMBLY 37 FIGURE 3. SCHEMATIC DIAGRAM OF CPEC PRINCIPLE 38 FIGURE 4. COMPARE THE STRENGTH OF NSSC/POPZ AND NSCC/POPZ SYSTEM. 40 FIGURE 5. PRELIMINARILY FUNCTIONAL TEST OF BETA-LACTAMASE 41 FIGURE 6. THE PRETREATMENT OF THE AGAROSE GEL AND IBIDI FOR TIME-LAPSE SHOOTING. 42 FIGURE 7. THE FLUORESCENT STRENGTH AND CAPPING CAPACITY OF THE M0052 AND M0050 DEGRADATION TAG. 44 FIGURE 8 FUNCTIONAL TEST OF THE DIVIVA::SFGFP::AMPR REPORTER PRODUCED BY NSCC/POPZ 46 FIGURE 9. DYNAMIC DISTRIBUTION OF THE REPORTER SIGNAL IN THE PERIOD OF CELL DIVISION. 47 FIGURE 10 THE TIME DIFFERENCE OF CELL DEATH BETWEEN TWO DAUGHTER CELLS. 48 FIGURE 11. THE DISTRIBUTION OF THE RED FLUORESCENCE SIGNAL IN THE TEV PROJECT. 50 FIGURE 12. FLUORESCENT EXPRESSION OF CPDR::MWASABI CONSTRUCT. 51 FIGURE 13. THE PLAN OF SIGNAL PEPTIDE OF THE BETA-LACTAMASE. 52 REFERENCE 53 | |
dc.language.iso | en | |
dc.title | 在大腸桿菌平台中實現功能分化 | zh_TW |
dc.title | Realization of Functional Differentiation in Escherichia coli | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 溫進德(Jin-Der Wen),吳?承(Hsuan-Chen WU) | |
dc.subject.keyword | 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, | zh_TW |
dc.subject.keyword | Synthetic biology,functional differentiation,cell polarization,PopZ,SpmX,DivIVA,asymmetric cell division,cell death, | en |
dc.relation.page | 53 | |
dc.identifier.doi | 10.6342/NTU201900988 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-14 | |
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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