合成基因迴路設計中，終止子大多扮演完全停止轉錄的角色，以避免上游轉錄單元干擾下游轉錄單元，然而，目前為止並沒有發現任何終止子俱有百分之百的終止效率，這將導致無法避免的干擾，使基因系統可能不符合設計者預期。此研究我們嘗試透過另一種全新的角度利用終止子，將終止子非百分之百的終止效率視為一種調控基因表現的方法，並且應用到基因迴路的設計，期望增加基因元件選擇的多樣性。設計迴路之前，我們首先對終止子進行量測與特性化分析，發現其終止效率並非定值，而是俱有非線性效應之特性，透過動態模型分析，我們觀察到此非線性特性提供基因迴路更多元的設計變化，我們也將設計的基因迴路轉殖到大腸桿菌中進行驗證，與動態模擬相符合，所設計之基因迴路利用終止子的非線性效應， 於大腸桿菌體內成功實現數個生物濾波器，包含高通濾波器以及帶通濾波器，結果顯示終止子作為調控元件的潛力。此外，此研究實現的帶通濾波器其設計複雜度較以往設計低，說明了終止子作為調控元件的優點，能夠使複雜的基因表達系統更容易達成。然而，終止子用於基因迴路的設計仍然處於初期階段，其中，對終止子的基本動態了解是應用到設計的重要關鍵，我們相信進一步探索終止子的複雜動態，對合成生物學的發展是重要且必須的。

Terminators, signaling the end of transcription process, are usually placed behind the last coding sequence of an operon to prevent interference between transcript units in most biologically synthetic systems. In this study, we seek to extend the usability of terminators in genetic system design by using terminators as regulatory genetic parts. Terminators with different impacts on their upstream and downstream genes are characterized in details via dynamic modeling to predict the behavior of the overall genetic system. Some nonlinear effects of terminators were observed in our terminator measurements. This potentially provides versatile regulation on gene expression. The genetic systems designed with regulatory terminators are predicted to behave like genetic filters through dynamic modeling in silico. In agreement with the simulations, genetic highpass and bandpass filters are successfully implemented in vitro, which demonstrates the potential of using terminators as regulatory parts. The genetic bandpass filter in this work is implemented through the interdependence between genetic parts, where the termination efficiency of terminator varies with the strength of upstream promoter. This design strategy of bandpass filter requires fewer base pairs than the conventional strategy of concatenating a highpass and lowpass filter. Our results show that novel utilization of terminators as regulatory parts can provide us a new perspective for efficient genetic circuit design. To move towards this direction, we believe that further exploration of the complicated dynamics of terminators is important for future advance in the development of synthetic biology.

[1] D. E. Cameron, C. J. Bashor, and J. J. Collins, "A brief history of synthetic biology," Nature Reviews Microbiology, vol. 12, pp. 381-390, May 2014.
[2] G. M. Church, M. B. Elowitz, C. D. Smolke, C. A. Voigt, and R. Weiss, "Realizing the potential of synthetic biology," Nature Reviews Molecular Cell Biology, vol. 15, pp. 289-294, Apr 2014.
[3] C. J. Paddon, P. J. Westfall, D. J. Pitera, K. Benjamin, K. Fisher, D. McPhee, et al., "High-level semi-synthetic production of the potent antimalarial artemisinin," Nature, vol. 496, pp. 528-+, Apr 25 2013.
[4] K. I. Ramalingam, J. R. Tomshine, J. A. Maynard, and Y. N. Kaznessis, "Forward engineering of synthetic bio-logical AND gates," Biochemical Engineering Journal, vol. 47, pp. 38-47, Dec 1 2009.
[5] A. Tamsir, J. J. Tabor, and C. A. Voigt, "Robust multicellular computing using genetically encoded NOR gates and chemical 'wires'," Nature, vol. 469, pp. 212-215, Jan 13 2011.
[6] M. B. E. Călin C. Guet, Weihong Hsing, Stanislas Leibler, "Combinatorial Synthesis of Genetic Networks," Science, vol. 296, pp. 1466-1470 2002.
[7] B. Wang, R. I. Kitney, N. Joly, and M. Buck, "Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology," Nature Communications, vol. 2, Oct 2011.
[8] J. Zhan, B. Ding, R. Ma, X. Ma, X. Su, Y. Zhao, et al., "Develop reusable and combinable designs for transcriptional logic gates," Molecular Systems Biology, vol. 6, Jul 2010.
[9] T. Danino, O. Mondragon-Palomino, L. Tsimring, and J. Hasty, "A synchronized quorum of genetic clocks," Nature, vol. 463, pp. 326-330, Jan 21 2010.
[10] M. B. Elowitz and S. Leibler, "A synthetic oscillatory network of transcriptional regulators," Nature, vol. 403, pp. 335-338, Jan 20 2000.
[11] J. Kim and E. Winfree, "Synthetic in vitro transcriptional oscillators," Molecular Systems Biology, vol. 7, Feb 2011.
[12] J. Stricker, S. Cookson, M. R. Bennett, W. H. Mather, L. S. Tsimring, and J. Hasty, "A fast, robust and tunable synthetic gene oscillator," Nature, vol. 456, pp. 516-U39, Nov 27 2008.
[13] S. Chen, H. Zhang, H. Shi, W. Ji, J. Feng, Y. Gong, et al., "Automated Design of Genetic Toggle Switches with Predetermined Bistability," Acs Synthetic Biology, vol. 1, pp. 284-290, Jul 2012.
[14] T. S. Gardner, C. R. Cantor, and J. J. Collins, "Construction of a genetic toggle switch in Escherichia coli," Nature, vol. 403, pp. 339-342, Jan 20 2000.
[15] T. S. Moon, E. J. Clarke, E. S. Groban, A. Tamsir, R. M. Clark, M. Eames, et al., "Construction of a Genetic Multiplexer to Toggle between Chemosensory Pathways in Escherichia coli," Journal of Molecular Biology, vol. 406, pp. 215-227, Feb 18 2011.
[16] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, and R. Weiss, "A synthetic multicellular system for programmed pattern formation," Nature, vol. 434, pp. 1130-1134, Apr 28 2005.
[17] D. Greber and M. Fussenegger, "An engineered mammalian band-pass network," Nucleic Acids Research, vol. 38, Oct 2010.
[18] S. Hooshangi, S. Thiberge, and R. Weiss, "Ultrasensitivity and noise propagation in a synthetic transcriptional cascade," Proceedings of the National Academy of Sciences of the United States of America, vol. 102, pp. 3581-3586, Mar 8 2005.
[19] T. Sohka, R. A. Heins, R. M. Phelan, J. M. Greisler, C. A. Townsend, and M. Ostermeier, "An externally tunable bacterial band-pass filter," Proceedings of the National Academy of Sciences of the United States of America, vol. 106, pp. 10135-10140, Jun 23 2009.
[20] J. J. Tabor, H. M. Salis, Z. B. Simpson, A. A. Chevalier, A. Levskaya, E. M. Marcotte, et al., "A Synthetic Genetic Edge Detection Program," Cell, vol. 137, pp. 1272-1281, Jun 26 2009.
[21] S. Atsumi, T. Hanai, and J. C. Liao, "Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels," Nature, vol. 451, pp. 86-U13, Jan 3 2008.
[22] Y.-X. Huo, K. M. Cho, J. G. L. Rivera, E. Monte, C. R. Shen, Y. Yan, et al., "Conversion of proteins into biofuels by engineering nitrogen flux," Nature Biotechnology, vol. 29, pp. 346-U160, Apr 2011.
[23] C. M. Immethun, A. G. Hoynes-O'Connor, A. Balassy, and T. S. Moon, "Microbial production of isoprenoids enabled by synthetic biology," Frontiers in Microbiology, vol. 4, Apr 4 2013.
[24] Z. Xie, L. Wroblewska, L. Prochazka, R. Weiss, and Y. Benenson, "Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells," Science, vol. 333, pp. 1307-1311, Sep 2 2011.
[25] R. Kwok, "FIVE HARD TRUTHS FOR SYNTHETIC BIOLOGY," Nature, vol. 463, pp. 288-290, Jan 21 2010.
[26] Y. Luo, J.-K. Lee, and H. Zhao, "Challenges and opportunities in synthetic biology for chemical engineers," Chemical Engineering Science, vol. 103, pp. 115-119, Nov 15 2013.
[27] D. E. a. T. F. K. J. Reshma P Shetty, "Engineering BioBrick vectors from BioBrick parts," Journal of Biological Engineering, 2008.
[28] H. M. Salis, E. A. Mirsky, and C. A. Voigt, "Automated design of synthetic ribosome binding sites to control protein expression," Nature Biotechnology, vol. 27, pp. 946-U112, Oct 2009.
[29] Y.-Y. Lee, C.-Y. Hsu, L.-J. Lin, C.-C. Chang, H.-C. Cheng, T.-H. Yeh, et al., "Systematic design methodology for robust genetic transistors based on I/O specifications via promoter-RBS libraries," Bmc Systems Biology, vol. 7, Oct 27 2013.
[30] J. M. Peters, A. D. Vangeloff, and R. Landick, "Bacterial Transcription Terminators: The RNA 3 '-End Chronicles," Journal of Molecular Biology, vol. 412, pp. 793-813, Oct 7 2011.
[31] J. Francis H.Martin and Ignacio Tinoco, "DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription," Nucleic Acids Research, vol. 8, 1980.
[32] I. G. a. E. Nudler, "The Mechanism of Intrinsic Transcription Termination," Molecular Cell, vol. 3, pp. 495–504, 1999.
[33] E. A. Lesnik, R. Sampath, H. B. Levene, T. J. Henderson, J. A. McNeil, and D. J. Ecker, "Prediction of rho-independent transcriptional terminators in Escherichia coli," Nucleic Acids Research, vol. 29, pp. 3583-3594, Sep 1 2001.
[34] C. Pichon and B. Felden, "Intergenic sequence inspector: searching and identifying bacterial RNAs," Bioinformatics, vol. 19, pp. 1707-1709, Sep 1 2003.
[35] C. L. Kingsford, K. Ayanbule, and S. L. Salzberg, "Rapid, accurate, computational discovery of Rho-independent transcription terminators illuminates their relationship to DNA uptake," Genome Biology, vol. 8, 2007 2007.
[36] P. P. Gardner, L. Barquist, A. Bateman, E. P. Nawrocki, and Z. Weinberg, "RNIE: genome-wide prediction of bacterial intrinsic terminators," Nucleic Acids Research, vol. 39, pp. 5845-5852, Aug 2011.
[37] R. A. Mooney and R. Landick, "Building a better stop sign: understanding the signals that terminate transcription," Nature Methods, vol. 10, pp. 618-619, Jul 2013.
[38] Y.-J. Chen, P. Liu, A. A. K. Nielsen, J. A. N. Brophy, K. Clancy, T. Peterson, et al., "Characterization of 582 natural and synthetic terminators and quantification of their design constraints," Nature Methods, vol. 10, pp. 659-+, Jul 2013.
[39] G. Cambray, J. C. Guimaraes, V. K. Mutalik, C. Lam, M. Quynh-Anh, T. Thimmaiah, et al., "Measurement and modeling of intrinsic transcription terminators," Nucleic Acids Research, vol. 41, pp. 5139-5148, May 2013.
[40] P. Siuti, J. Yazbek, and T. K. Lu, "Synthetic circuits integrating logic and memory in living cells," Nature Biotechnology, vol. 31, pp. 448-+, May 2013.
[41] J. Bonnet, P. Yin, M. E. Ortiz, P. Subsoontorn, and D. Endy, "Amplifying Genetic Logic Gates," Science, vol. 340, pp. 599-603, May 3 2013.
[42] B. F. Pfleger, D. J. Pitera, C. D Smolke, and J. D. Keasling, "Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes," Nature Biotechnology, vol. 24, pp. 1027-1032, Aug 2006.
[43] R. S. P. a. T. L. Keith L. Steward, "Transcription-frequency-dependent modulation of an attenuator in a ribosomal protein–RNA polymerase operon requires an upstream site," Microbiology, 1997.
[44] M. J. C. Alice P.W. Telesnitsky, "Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites," Journal of Molecular Biology, vol. 205, pp. 315-330, 20 January 1989 1989.
[45] H. A. H Abe, "Differential contributions of two elements of rho-independent terminator to transcription termination and mRNA stabilization," Biochimie, vol. 78, pp. 1035-1042, 1996.
[46] K. A. Curran, A. S. Karim, A. Gupta, and H. S. Alper, "Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications," Metab Eng, vol. 19, pp. 88-97, Sep 2013.
[47] T. Nojima, A. C. Lin, T. Fujii, and I. Endo, "Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants," Analytical Sciences, vol. 21, pp. 1479-1481, Dec 2005.