利用合成生物學的設計方法建立使細菌搜尋特定濃度小分子之基因電路

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研究生：	田欣民 Tien, Hsin Min
論文名稱：	利用合成生物學的設計方法建立使細菌搜尋特定濃度小分子之基因電路 Engineering bacteria to search for specific concentrations of molecules by a systematic synthetic biology design method
指導教授：	陳博現 Chen, Bor Sen
口試委員:	沈若樸 Shen, Roa Pu 蘭宜錚 Lan, Ethan
學位類別：	碩士 Master
系所名稱：	電機資訊學院 - 電機工程學系 Department of Electrical Engineering
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	78
中文關鍵詞：	合成生物、CheY D13KY106W
外文關鍵詞：	synthetic biology, CheY D13KY106W
相關次數：	點閱：2 下載：0
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細菌利用複雜的訊號傳遞系統控制鞭毛運動以在充滿各種物質的環境內尋找適當生存住所。藉由影響細菌的訊號傳遞路徑使細菌尋找特定的化學物質有機會開創嶄新的應用在生物醫學、環境復育等領域。在本研究中，我們利用合成生物學的方法建立可使細菌尋找特定化學物質並定位在該物質分布區域的基因電路。此外，藉由更換電路內不同靈敏度的「剎車」元件，細菌可被設計用於定位於特定濃度的區域。利用洋菜膠菌落擴散實驗(swarm assay)(定性)及微流體技術(microfluidic technique)(定量)做測試，各別「剎車」元件的特性可被定義，並以一個數學模型表示。我們更進一步建立一個可預測「剎車」元件特性的數學模型。利用此模型，在不需要對元件各別測試的情況下，巨大的元件庫可被建立並用於不同的偵測濃度。最後，我們提出一個設計流程供讀者參考。依照此流程，讀者可藉由挑選適當的「剎車」元件以設計出可使細菌搜尋特定濃度之特定物質的基因電路。另外，依照簡單的步驟，讀者也可自行建立適用於其他培養環境、啟動子系統或不同菌種的專屬元件庫。我們希望隨著合成生物學領域的發展，本研究中的設計方法可被應用於其他具有特定功能的基因電路。藉此，我們預期可拓展合成生物學在環境複育、奈米科技以及醫學等領域之應用。

Bacteria navigate the environment full of various chemicals to seek favorable places for surviving by controlling the flagella’s rotation using a complicated signal transduction pathway. By influencing the pathway, bacteria can be engineered to search for specific molecules, which has great potential for application to biomedicine and bioremediation. In this study, genetic circuits were constructed to make bacteria search for specific molecule and locate by the synthetic biology method. In addition, by replacing the “brake component” in the synthetic circuit with ones with specific sensitivities, the bacteria can be engineered to locate in areas containing specific concentrations of the molecule. Measured by swarm assays qualitatively and microfluidic technique quantitatively, the characteristic of each “brake component” was identified and represented by a mathematical model. Furthermore, we established another mathematical model to anticipate the characteristic of the “brake component”. Based on this model, an abundant component library can be established to provide selection for different searching conditions without identifying all the components one by one. Finally, a systematic design procedure was proposed. Following the systematic procedure, one can design a genetic circuit to make bacteria search for specific molecule of specific concentration by selecting the most adequate “brake component” in the library. Moreover, following simple procedures, one can also establish one’s exclusive component library suitable for other cultivated environment, promoter systems or bacterial strains.

摘 要……………………………………………………………………………..………...    i
Abstract……………………………………………………………………………………..    ii
致謝…………………………………………………………………………………………iii
Content…………………………………………………………………………….…..........    iv
List of Figures……………………………………………………………………….....…...    v
List of Tables…………………………………………………………………………….....    v
Introduction……………………………………………………………………………........    1
Construction of synthetic genetic circuit in response to different concentrations of AH…..    7
  2.1 Construction and verification of the basic circuit………………...………………….    8
  2.2 Improvement of the circuits’ performance by constructing the “brake components”
Library………………………………………………………………………………..    10
  2.3 Verification of the function of the components by swarm assays…………………...    11
Measurement and identification of the “brake components”……………………………….    12
  3.1 Methods for measuring bacteria population diffusing rate…………………………..    13
  3.2 Measurement of the “brake components”……………………………………………    15
  3.3 Constructing a mathematical model to identify the “brake components”……….......    17
  3.4 Enrichment of the “brake components” library by mutations…………………….....    20
Constructing mathematical models for simulating the behavior of the synthetic circuit…..    22
  4.1 Dynamic model of the synthetic circuit……………………………………………...    23
  4.2 Identification of the diffusion rate of the bacterial population at all AHL
Concentrations……………………………………………………………………….    25
  4.3 Constructing a mathematical model to simulate the characteristic of the “brake 
components” based on components’ composition…………………………………...    27
  4.4 Design strategy for the synthetic circuit……………………………………………..    31
Discussion…………………………………………………………………………………..    34
Conclusion………………………………………………………………………………….    36
Reference…………………………………………………………………………………...    37
Figures………………………………………………………………………………………    40
Tables……………………………………………………………………………………….    52
Supplementary Information………………………………………………………………...    55
A.    Supplementary Tables………………………………………………………………..    55
B.    Supplementary Figures………………………………………………………………    58
C.    Materials and Methods……………………………………………………………….    74


List of Figures
Figure 1.    The basic design scheme of the synthetic circuit…………………………………    40
Figure 2.    Verifying the performance of the synthetic circuit in Figure 1 in response to
    AHL……………………………………………………………………………….    41
Figure 3.     Improving the basic circuit by constructing a series of “brake components”…….    42
Figure 4.     Verification of the brake components by swarm assays…………………………..    43
Figure 5.     The methodology for measuring bacterial population diffusion rate……………..    44
Figure 6.     Comparing the effect of each brake component in response to different AHL        concentrations……………………………………………………………………..    45
Figure 7.     Representation of components’ characteristic by Eq. (5)…………………………    46
Figure 8.     Simulating “stimulus vs. CAL” and “stimulus vs. diffusion rate” based on the 
    dynamic model…………………………………………………………………….    47
Figure 9.     Assumptions for establishing the part-based mathematical model………………..    48
Figure 10.    Comparison between the simulated characteristic of the components and the experimental results……………………………………………………………….    49
Figure 11.    Flow chart for designing a synthetic circuit which can search for a specific
    stimulus of a specific concentration……………………………………………….    50
Figure 12.    The simulated and experimental result of the component chosen to search for 
    10-9 M AHL………………………………………………………………………..    51


List of Tables
Table 1.     Information of the identified “brake components”………………………………..    52
Table 2.     Parameters in dynamic model of the PluxB0034 promoter-RBS pair…………….    53
Table 3.     Members and parameters of the “parts library”…………………………………...    54

                                

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