銅離子在人體內扮演著重要的角色，例如參與呼吸作用電子傳遞鏈以及一些神經訊息的傳遞。然而銅離子濃度過高也會導致一些疾病，例如威爾森氏症、帕金森氏症。苯乙胺是神經傳導物質，苯乙酸是其代謝產物，一些疾病如苯丙酮尿症和精神分裂症患者的尿液中可偵測到過量的苯乙胺和苯乙酸。本研究期望能提供以微生物系統作為感測的新方法。偵測銅離子方面我們表達耐金屬貪銅菌金離子調控組CueR的啟動子PcopZ和銅離子調控組CopSR的啟動子PcopA，偵測苯乙胺和苯乙酸方面同時表達大腸桿菌的單胺類調控組Mao、苯乙酸代謝調控組Paa，兩者分別接上紅色螢光蛋白與綠色螢光蛋白作為雙重訊號輸出。藉由檢測紅色螢光與綠色螢光之訊號，我們設計的感測器對銅離子、苯乙胺、苯乙酸的偵測具有高度專一性和靈敏性。

Copper ions act as an important role in our body, involving cellular respiration and signal transduction. Some neurodegenerative diseases, such as Wilson diseases and Alzheimer’s disease, are resulted from high copper ion concentration. β-Phenylethylamine (PEA) is an important neurotransmitter in our body and phenylacetic acid (PA) is its metabolite. Patients with special diseases such as phenylketonuria and paranoid schizophrenia have high concentration of PEA and PA in body fluids. We constructed two dual signal whole-cell biosensors with the expression GFP and RFP for detection copper ions, PEA, and PA. First, we used promoter PcopZ and PcopA in Cupriavidus metallidurans to exclude the interference caused by other metal ions. Second, we used PEA and PA degradation operon in Escherichia coli to detect PEA and PA simultaneously. By monitoring RFP and GFP fluorescence, we both had good sensitivity and selectivity for detection of Cu2+, PEA, and PA.

1. Kovach, M. E., et al., Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 1995, 166 (1), 175-176.
2. de Lorenzo, V., et al., Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. Journal of Bacteriology 1990, 172 (11), 6568-6572.
3. Contag, C. H. andBachmann, M. H., Advances in In Vivo Bioluminescence Imaging of Gene Expression. Annual Review of Biomedical Engineering 2002, 4 (1), 235-260.
4. Kushwaha, M. andSalis, H. M., A portable expression resource for engineering cross-species genetic circuits and pathways. Nature communications 2015, 6, 7832.
5. Watstein, D. M. andStyczynski, M. P., Development of a Pigment-Based Whole-Cell Zinc Biosensor for Human Serum. ACS Synthetic Biology 2018, 7 (1), 267-275.
6. Ostrov, N., et al., A modular yeast biosensor for low-cost point-of-care pathogen detection. Science Advances 2017, 3 (6), e1603221.
7. Turner, K., et al., Transcriptional regulatory proteins as biosensing tools. Chemical Communications 2017, 53 (51), 6820-6823.
8. Shannon, C. E., A symbolic analysis of relay and switching circuits. Electrical Engineering 1938, 57 (12), 713-723.
9. Weiss, R., Homsy, G.E. & Knight Jr, T.F., Toward in vivo digital circuits. Proceedings of the DIMACS Workshop on Evolution as Computation 1999.
10. Strack, G., et al., Biocomputing Security System:  Concatenated Enzyme-Based Logic Gates Operating as a Biomolecular Keypad Lock. Journal of the American Chemical Society 2008, 130 (13), 4234-4235.
11. Moshe, M., et al., Sensing of UO22+ and Design of Logic Gates by the Application of Supramolecular Constructs of Ion-Dependent DNAzymes. Nano Letters 2009, 9 (3), 1196-1200.
12. Wang, B., et al., Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature communications 2011, 2, 508.
13. Collmer, A., et al., <em>Pseudomonas syringae</em> Hrp type III secretion system and effector proteins. Proceedings of the National Academy of Sciences 2000, 97 (16), 8770.
14. Bereza-Malcolm, L. T., et al., Environmental Sensing of Heavy Metals Through Whole Cell Microbial Biosensors: A Synthetic Biology Approach. ACS Synthetic Biology 2015, 4 (5), 535-546.
15. Wang, B., et al., A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals. Biosensors & bioelectronics 2013, 40 (1), 368-376.
16. Mergeay, M., et al., Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. FEMS microbiology reviews 2003, 27 (2-3), 385-410.
17. Stock, A. M., et al., Two-Component Signal Transduction. Annual Review of Biochemistry 2000, 69 (1), 183-215.
18. Monchy, S., et al., Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34. Microbiology 2006, 152 (6), 1765-1776.
19. Pohlmann, A., et al., Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 2006, 24, 1257.
20. Schlegel, H. G., et al., Formation and Utilization of Poly-β-Hydroxybutyric Acid by Knallgas Bacteria (Hydrogenomonas). Nature 1961, 191, 463.
21. Luzier, W. D., Materials derived from biomass/biodegradable materials. Proceedings of the National Academy of Sciences 1992, 89 (3), 839.
22. Das, S., et al., Genetic basis and importance of metal resistant genes in bacteria for bioremediation of contaminated environments with toxic metal pollutants. Applied microbiology and biotechnology 2016, 100 (7), 2967-2984.
23. Truong, K. andIkura, M., The use of FRET imaging microscopy to detect protein–protein interactions and protein conformational changes in vivo. Current Opinion in Structural Biology 2001, 11 (5), 573-578.
24. Johnsson, N. andVarshavsky, A., Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 1994, 91 (22), 10340-10344.
25. Remy, I. andMichnick, S. W., A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nature methods 2006, 3 (12), 977-979.
26. Galarneau, A., et al., Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nature biotechnology 2002, 20 (6), 619-622.
27. Ghosh, I., et al., Antiparallel Leucine Zipper-Directed Protein Reassembly:  Application to the Green Fluorescent Protein. Journal of the American Chemical Society 2000, 122 (23), 5658-5659.
28. Landschulz, W. H., et al., The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science (New York, N.Y.) 1988, 240 (4860), 1759.
29. Wall, M. E. andWani, M. C., Paclitaxel: From Discovery to Clinic. In Taxane Anticancer Agents, American Chemical Society: 1994; Vol. 583, pp 18-30.
30. Kantarjian Hagop, M., et al., Homoharringtonine. Cancer 2001, 92 (6), 1591-1605.
31. Reineke, W., Development of hybrid strains for the mineralization of chloroaromatics by patchwork assembly. Annual review of microbiology 1998, 52, 287-331.
32. Díaz, E., et al., Biodegradation of Aromatic Compounds by Escherichia coli. Microbiology and Molecular Biology Reviews 2001, 65 (4), 523-569.
33. Hatfield, E. andSprecher, S., Measuring passionate love in intimate relationships. Journal of Adolescence 1986, 9 (4), 383-410.
34. Volkow, N. D., et al., Therapeutic doses of oral methylphenidate significantly increase extracellular dopamine in the human brain. The Journal of neuroscience : the official journal of the Society for Neuroscience 2001, 21 (2), Rc121.
35. Taylor, F. B. andRusso, J., Comparing Guanfacine and Dextroamphetamine for the Treatment of Adult Attention-Deficit/Hyperactivity Disorder. Journal of Clinical Psychopharmacology 2001, 21 (2).
36. Hoch, P. H., et al., EFFECTS OF MESCALINE AND LYSERGIC ACID (d-LSD-25). American Journal of Psychiatry 1952, 108 (8), 579-584.
37. Green, A. R., et al., The Pharmacology and Clinical Pharmacology of 3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”). Pharmacological Reviews 2003, 55 (3), 463.
38. Janssen, P. A., et al., Does phenylethylamine act as an endogenous amphetamine in some patients? The international journal of neuropsychopharmacology 1999, 2 (3), 229-240.
39. Irsfeld, M., et al., β-phenylethylamine, a small molecule with a large impact. WebmedCentral 2013, 4 (9), 4409.
40. Oldendorf, W. H., Brain uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. The American journal of physiology 1971, 221 (6), 1629-1639.
41. Baker, G. B., et al., Phenylethylaminergic mechanisms in attention-deficit disorder. Biological psychiatry 1991, 29 (1), 15-22.
42. Xie, Z., et al., Modulation of Monoamine Transporters by Common Biogenic Amines via Trace Amine-Associated Receptor 1 and Monoamine Autoreceptors in Human Embryonic Kidney 293 Cells and Brain Synaptosomes. Journal of Pharmacology and Experimental Therapeutics 2008, 325 (2), 629.
43. Halász, A., et al., Biogenic amines and their production by microorganisms in food. Trends in Food Science & Technology 1994, 5 (2), 42-49.
44. Diamond, A., et al., Prefrontal Cortex Cognitive Deficits in Children Treated Early and Continuously for PKU. Monographs of the Society for Research in Child Development 1997, 62 (4), i-206.
45. Chen, P.-H., et al., Development of a pigment-based whole-cell biosensor for the analysis of environmental copper. RSC Advances 2017, 7 (47), 29302-29305.
46. Suzanne, L., et al., Engineered Bacteria Based Biosensors for Monitoring Bioavailable Heavy Metals. Electroanalysis 2002, 14 (1), 35-42.
47. Ng, S. P., et al., Identification of a copper-responsive promoter and development of a copper biosensor in the soil bacterium Achromobacter sp. AO22. World Journal of Microbiology and Biotechnology 2012, 28 (5), 2221-2228.
48. Shen, Q., et al., A simple “clickable” biosensor for colorimetric detection of copper(II) ions based on unmodified gold nanoparticles. Biosensors and Bioelectronics 2013, 41, 663-668.
49. Chen, W., et al., Fluorescent gold nanoparticles-based fluorescence sensor for Cu2+ ions. Chemical Communications 2009, (13), 1736-1738.
50. Chan, Y.-H., et al., Ultrasensitive Copper(II) Detection Using Plasmon-Enhanced and Photo-Brightened Luminescence of CdSe Quantum Dots. Analytical Chemistry 2010, 82 (9), 3671-3678.
51. Zheng, Y., et al., A dansylated peptide for the selective detection of copper ions. Chemical Communications 2002, (20), 2350-2351.
52. Salaün, P. andvan den Berg, C. M. G., Voltammetric Detection of Mercury and Copper in Seawater Using a Gold Microwire Electrode. Analytical Chemistry 2006, 78 (14), 5052-5060.
53. Aigner, M., et al., Amperometric biosensor for total monoamines using a glassy carbon paste electrode modified with human monoamine oxidase B and manganese dioxide particles. Microchimica Acta 2015, 182 (5), 925-931.
54. Han, J., et al., Water-Soluble Poly(p-aryleneethynylene)s: A Sensor Array Discriminates Aromatic Carboxylic Acids. ACS Applied Materials & Interfaces 2016, 8 (31), 20415-20421.
55. F., B. U. H., Poly(aryleneethynylene)s. Macromolecular Rapid Communications 2009, 30 (9‐10), 772-805.
56. Dierckx, S., et al., Design and Construction of a Whole Cell Bacterial 4-Hydroxyphenylacetic Acid and 2-Phenylacetic Acid Bioassay. Frontiers in Bioengineering and Biotechnology 2015, 3, 88.
57. Goswami, S., et al., Recognition of Carboxylate Anions and Carboxylic Acids by Selenium-Based New Chromogenic Fluorescent Sensor: A Remarkable Fluorescence Enhancement of Hindered Carboxylates. Organic Letters 2009, 11 (19), 4350-4353.
58. Campbell, R. E., et al., A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences 2002, 99 (12), 7877.
59. Zhang, G., et al., An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical and Biophysical Research Communications 1996, 227 (3), 707-711.
60. Guo, K.-H., et al., Determination of Gold Ions in Human Urine Using Genetically Engineered Microorganisms on a Paper Device. ACS Sensors 2018, 3 (4), 744-748.
61. Fernandez, C., et al., Insights on the regulation of the phenylacetate degradation pathway from Escherichia coli. Environmental microbiology reports 2014, 6 (3), 239-250.
62. Lin, Y.-K. andYeh, Y.-C., Dual-Signal Microbial Biosensor for the Detection of Dopamine without Inference from Other Catecholamine Neurotransmitters. Analytical Chemistry 2017, 89 (21), 11178-11182.
63. Yeh, Y.-C., et al., Functionalizing bacterial cell surfaces with a phage protein. Chemical Communications 2013, 49 (9), 910-912.
64. Bi, C., et al., Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production. Microbial Cell Factories 2013, 12 (1), 107.