簡易檢索 / 詳目顯示

研究生: 陳鴻暉
Chen, Hung-Hui
論文名稱: 探討強健且外部可調式的生物濾波器在大腸桿菌內的作用機制
Study on the mechanism of robust and externally tunable biological filters in Escherichia coli
指導教授: 徐邦達
Hsu, Ban-Dar
口試委員: 陳博現
李曉青
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年: 2013
畢業學年度: 101
語文別: 中文
論文頁數: 52
中文關鍵詞: 合成生物學基因電路生物濾波器
外文關鍵詞: synthetic biology, genetic circuit, biological filter
相關次數: 點閱:3下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 合成生物學的目的在於以可替換的標準化元件模擬生物行為,及建構具有特定功能的新穎人工生物系統。在本篇研究中,我們運用了以基因演算法 (genetic algorithms, GA) 為基礎的搜尋方法,從充分特性化的啟動子 - 核糖體結合位元件庫中,有效地尋找適當元件,並在大腸桿菌內建構一套具有特定輸入/輸出響應,強健且可受外部調控的合成生物濾波器裝置。基因電路的閾值可受控制型誘導物的濃度調節。
    綠螢光蛋白 (GFP) 在本實驗的基因電路中被用來做為報導蛋白,可使用酵素免疫分析儀 (ELISA reader) 偵測其表現量。我們證實了生物濾波器在生物體內的基因表現模式,對應輸入的化學物質濃度梯度分別展示出低通和高通的特性。在未來,我們可挑選不同具有相異動態活性的啟動子 - 核糖體結合位元件,精確調整輸入/輸出響應的閾值,使實驗結果更能吻合模型預測。生物濾波器可在發育生物學中的形式生成,生物感測器,以及治療癌症上提供相當廣泛的應用。


    Synthetic biology aims both to employ standard interchangeable components to mimic biological behavior, and to construct novel artificial biological systems with prescribed functions. In this study, the GA-based searching method was used to efficiently select an adequate set of promoter-RBS components from the well-characterized promoter-RBS libraries, and robust externally tunable synthetic biological filter devices with a specified input/output (I/O) response were built in Escherichia coli. The thresholds of these gene circuits could be tuned by adjusting the concentration of the control inducer.
    The green fluorescent protein (GFP) was employed as the reporter protein in the gene circuit, of which expression levels were measured using an ELISA reader. The gene expression patterns of these biological filters exhibited low-pass and high-pass like characteristics respectively in response to an input chemical gradient in vivo. In the future, different promoter-RBS components with different kinetic activities may be selected to precisely tune the threshold of the I/O response of filters, thereby making the experimental results more closely fit the model prediction. The biological filters may provide a wide variety of applications in pattern formation, biosensor, and cancer therapy.

    摘要 1 前言 3 材料與方法 8 結果 16 討論 22 圖表 27 表格 44 附錄 45 參考文獻 48

    1. Benner, S.A., and Sismour, A.M. (2005). Synthetic biology. Nat Rev Genet 6, 533-543.
    2. Andrianantoandro, E., Basu, S., Karig, D.K., and Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol 2, 2006 0028.
    3. Canton, B., Labno, A., and Endy, D. (2008). Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-793.
    4. Shetty, R.P., Endy, D., and Knight, T.F., Jr. (2008). Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2, 5.
    5. Anderson, J.C., Dueber, J.E., Leguia, M., Wu, G.C., Goler, J.A., Arkin, A.P., and Keasling, J.D. (2010). BglBricks: A flexible standard for biological part assembly. J Biol Eng 4, 1.
    6. Win, M.N., and Smolke, C.D. (2008). Higher-order cellular information processing with synthetic RNA devices. Science 322, 456-460.
    7. Leisner, M., Bleris, L., Lohmueller, J., Xie, Z., and Benenson, Y. (2010). Rationally designed logic integration of regulatory signals in mammalian cells. Nat Nanotechnol 5, 666-670.
    8. Regot, S., Macia, J., Conde, N., Furukawa, K., Kjellen, J., Peeters, T., Hohmann, S., de Nadal, E., Posas, F., and Sole, R. (2011). Distributed biological computation with multicellular engineered networks. Nature 469, 207-211.
    9. Tamsir, A., Tabor, J.J., and Voigt, C.A. (2011). Robust multicellular computing using genetically encoded NOR gates and chemical 'wires'. Nature 469, 212-215.
    10. Wang, B., Kitney, R.I., Joly, N., and Buck, M. (2011). Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Commun 2, 508.
    11. Auslander, S., Auslander, D., Muller, M., Wieland, M., and Fussenegger, M. (2012). Programmable single-cell mammalian biocomputers. Nature 487, 123-127.
    12. Lu, T.K., Khalil, A.S., and Collins, J.J. (2009). Next-generation synthetic gene networks. Nat Biotechnol 27, 1139-1150.
    13. Purnick, P.E., and Weiss, R. (2009). The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol 10, 410-422.
    14. Khalil, A.S., and Collins, J.J. (2010). Synthetic biology: applications come of age. Nat Rev Genet 11, 367-379.
    15. Elowitz, M.B., and Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338.
    16. Gardner, T.S., Cantor, C.R., and Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339-342.
    17. Atkinson, M.R., Savageau, M.A., Myers, J.T., and Ninfa, A.J. (2003). Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in Escherichia coli. Cell 113, 597-607.
    18. Kramer, B.P., Viretta, A.U., Daoud-El-Baba, M., Aubel, D., Weber, W., and Fussenegger, M. (2004). An engineered epigenetic transgene switch in mammalian cells. Nat Biotechnol 22, 867-870.
    19. Basu, S., Gerchman, Y., Collins, C.H., Arnold, F.H., and Weiss, R. (2005). A synthetic multicellular system for programmed pattern formation. Nature 434, 1130-1134.
    20. Stricker, J., Cookson, S., Bennett, M.R., Mather, W.H., Tsimring, L.S., and Hasty, J. (2008). A fast, robust and tunable synthetic gene oscillator. Nature 456, 516-519.
    21. Friedland, A.E., Lu, T.K., Wang, X., Shi, D., Church, G., and Collins, J.J. (2009). Synthetic gene networks that count. Science 324, 1199-1202.
    22. Sohka, T., Heins, R.A., and Ostermeier, M. (2009). Morphogen-defined patterning of Escherichia coli enabled by an externally tunable band-pass filter. J Biol Eng 3, 10.
    23. Sohka, T., Heins, R.A., Phelan, R.M., Greisler, J.M., Townsend, C.A., and Ostermeier, M. (2009). An externally tunable bacterial band-pass filter. Proc Natl Acad Sci U S A 106, 10135-10140.
    24. Tigges, M., Marquez-Lago, T.T., Stelling, J., and Fussenegger, M. (2009). A tunable synthetic mammalian oscillator. Nature 457, 309-312.
    25. Danino, T., Mondragon-Palomino, O., Tsimring, L., and Hasty, J. (2010). A synchronized quorum of genetic clocks. Nature 463, 326-330.
    26. Greber, D., and Fussenegger, M. (2010). An engineered mammalian band-pass network. Nucleic Acids Res 38, e174.
    27. Quintero, A., Garcia, S., Guevara, C., Rincon, C., Ospina, C., Guevara, P., and Cuero, R. (2007). A microbial biosensor device for iron detection under UV irradiation. Synthetic Biology, IET 1, 71-73.
    28. Kampf, M.M., Engesser, R., Busacker, M., Horner, M., Karlsson, M., Zurbriggen, M.D., Fussenegger, M., Timmer, J., and Weber, W. (2012). Rewiring and dosing of systems modules as a design approach for synthetic mammalian signaling networks. Mol Biosyst 8, 1824-1832.
    29. Wang, B., Barahona, M., and Buck, M. (2013). A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals. Biosens Bioelectron 40, 368-376.
    30. de Lorenzo, V. (2008). Systems biology approaches to bioremediation. Curr Opin Biotechnol 19, 579-589.
    31. Lee, S.K., Chou, H., Ham, T.S., Lee, T.S., and Keasling, J.D. (2008). Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19, 556-563.
    32. Weber, W., and Fussenegger, M. (2009). The impact of synthetic biology on drug discovery. Drug Discov Today 14, 956-963.
    33. Ruder, W.C., Lu, T., and Collins, J.J. (2011). Synthetic biology moving into the clinic. Science 333, 1248-1252.
    34. Keasling, J.D. (2012). Synthetic biology and the development of tools for metabolic engineering. Metab Eng 14, 189-195.
    35. Weber, W., and Fussenegger, M. (2012). Emerging biomedical applications of synthetic biology. Nat Rev Genet 13, 21-35.
    36. Kelly, J.R., Rubin, A.J., Davis, J.H., Ajo-Franklin, C.M., Cumbers, J., Czar, M.J., de Mora, K., Glieberman, A.L., Monie, D.D., and Endy, D. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 3, 4.
    37. Cheng, A.A., and Lu, T.K. (2012). Synthetic biology: an emerging engineering discipline. Annu Rev Biomed Eng 14, 155-178.
    38. Ellis, T., Wang, X., and Collins, J.J. (2009). Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotechnol 27, 465-471.
    39. de Jong, H. (2002). Modeling and simulation of genetic regulatory systems: a literature review. J Comput Biol 9, 67-103.
    40. Chandran, D., Copeland, W.B., Sleight, S.C., and Sauro, H.M. (2008). Mathematical modeling and synthetic biology. Drug Discovery Today: Disease Models 5, 299-309.
    41. Zheng, Y., and Sriram, G. (2010). Mathematical modeling: bridging the gap between concept and realization in synthetic biology. J Biomed Biotechnol 2010, 541609.
    42. Guido, N.J., Wang, X., Adalsteinsson, D., McMillen, D., Hasty, J., Cantor, C.R., Elston, T.C., and Collins, J.J. (2006). A bottom-up approach to gene regulation. Nature 439, 856-860.
    43. Cox, R.S., 3rd, Surette, M.G., and Elowitz, M.B. (2007). Programming gene expression with combinatorial promoters. Mol Syst Biol 3, 145.
    44. Salis, H.M., Mirsky, E.A., and Voigt, C.A. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27, 946-950.
    45. Yokobayashi, Y., Weiss, R., and Arnold, F.H. (2002). Directed evolution of a genetic circuit. Proc Natl Acad Sci U S A 99, 16587-16591.
    46. Tyo, K.E., Nevoigt, E., and Stephanopoulos, G. (2011). Directed evolution of promoters and tandem gene arrays for customizing RNA synthesis rates and regulation. Methods Enzymol 497, 135-155.
    47. Cobb, R.E., Si, T., and Zhao, H. (2012). Directed evolution: an evolving and enabling synthetic biology tool. Curr Opin Chem Biol 16, 285-291.
    48. Wu, C.H., Lee, H.C., and Chen, B.S. (2011). Robust synthetic gene network design via library-based search method. Bioinformatics 27, 2700-2706.
    49. Tsien, R.Y. (1998). The green fluorescent protein. Annu Rev Biochem 67, 509-544.
    50. Wang, B., and Buck, M. (2012). Customizing cell signaling using engineered genetic logic circuits. Trends Microbiol 20, 376-384.
    51. Levskaya, A., Chevalier, A.A., Tabor, J.J., Simpson, Z.B., Lavery, L.A., Levy, M., Davidson, E.A., Scouras, A., Ellington, A.D., Marcotte, E.M., et al. (2005). Synthetic biology: engineering Escherichia coli to see light. Nature 438, 441-442.
    52. Weber, W., Marty, R.R., Link, N., Ehrbar, M., Keller, B., Weber, C.C., Zisch, A.H., Heinzen, C., Djonov, V., and Fussenegger, M. (2003). Conditional human VEGF-mediated vascularization in chicken embryos using a novel temperature-inducible gene regulation (TIGR) system. Nucleic Acids Res 31, e69.
    53. Lang, C., Pollithy, A., and Schuler, D. (2009). Identification of promoters for efficient gene expression in Magnetospirillum gryphiswaldense. Appl Environ Microbiol 75, 4206-4210.
    54. Tabor, J.J., Salis, H.M., Simpson, Z.B., Chevalier, A.A., Levskaya, A., Marcotte, E.M., Voigt, C.A., and Ellington, A.D. (2009). A synthetic genetic edge detection program. Cell 137, 1272-1281.
    55. Prindle, A., Samayoa, P., Razinkov, I., Danino, T., Tsimring, L.S., and Hasty, J. (2012). A sensing array of radically coupled genetic 'biopixels'. Nature 481, 39-44.
    56. Callura, J.M., Cantor, C.R., and Collins, J.J. (2012). Genetic switchboard for synthetic biology applications. Proc Natl Acad Sci U S A 109, 5850-5855.
    57. Paitan, Y., Biran, I., Shechter, N., Biran, D., Rishpon, J., and Ron, E.Z. (2004). Monitoring aromatic hydrocarbons by whole cell electrochemical biosensors. Anal Biochem 335, 175-183.
    58. Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R., and Benenson, Y. (2011). Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333, 1307-1311.

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)

    QR CODE