本研究使用微生物抵抗銅離子的調控系統，利用基因重組技術設計量測銅離子的全細胞生物感測器。本研究分別使用青枯桿菌 (Ralstonia eutropha) 中屬於 MerR 家族的 cue 基因調控組，以及耐金屬貪銅菌 (Cupriavidus metallidurans) 中屬於二元調節系統的 cop 基因調控組，以紅色螢光蛋白作為訊號輸出的報導基因來建構質體，設計出不同的銅離子生物感測器。其中，cueR 生物感測器除了能夠量測銅離子之外，在適當的前處理下還能分別量測銀離子與金離子。根據世界衛生組織所公布的飲用水水質準則，飲用水中銅離子的含量不得超過 2 mg/L (31 μM)，本研究所設計的 cueR 生物感測器以耐金屬貪銅菌作為質體之宿主時，量測銅離子的最低偵測極限為 25.54 μM，能夠量測飲用水中銅離子是否超標。而在 copSR 生物感測器的實驗中，嘗試使用不同的 cop 啟動子，建構出多種 copSR 生物感測器。也嘗試表現紫茉莉的 4,5-多巴雙加氧酶 (DOPA 4,5-Dioxygenase of Mirabilis jalapa, MjDOD)，透過添加 L-多巴 (L-3,4-dihydroxyphenylalanine, L-DOPA) 催化產生出甜菜黃色素 (betaxanthin)，以色素的生成作為輸出訊號，進而縮短檢測時間。此外，我們嘗試在不同來源的水樣品中額外添加銅離子進行量測，證明本研究設計的 copSR 生物感測器不會受到水中其他雜質干擾而影響銅離子量測。本研究設計出多種銅離子生物感測器，隨著每種生物感測器偵測範圍不同，可望運用在不同的需求上。

In this study, we used recombinant DNA technology to develop whole-cell biosensors for the detection of copper ions. The bacterial copper resistance regulons of MerR-family and two-component system (Ralstonia eutropha cue regulon and Cupriavidus merallidurans cop regulon, respectively) were used to regulate the expression of rfp (red fluorescence protein) reporter gene for the construction of whole-cell biosensors. Our results demonstrated that cueR-based biosensor could detect copper, silver, and gold ions under appropriate pretreatments. When using C. metallidurans as a host cell, the limit of detection for copper was 25.54 μM. According to World Health Organization (WHO) guidelines, the recommended value of copper in drinking water is below 2 mg/L (31 μM). The cueR-based copper biosensor designed in this study can successfully detect whether drinking water contains excessive copper ions. In the case of copSR-based biosensors, different copSR-based biosensors were constructed by screening various cop promoters. In order to reduce the detection times, we expressed DOPA 4,5-Dioxygenase of Mirabilis jalapa (MjDOD) which used L-DOPA (L-3,4-dihydroxyphenylalanine) as a substrate to produce yellow fluorescent pigments, betaxanthins. Furthermore, we added known concentrations of copper ions to real water samples, such as tap water and pond water. Our copSR whole-cell biosensor showed no significant sample matrix effect. With various detection ranges and different detection limits, copper biosensors in this study can provide applications with different windows of detection.

1. Halliwell, B.; Gutteridge, J. M., Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984, 219, 1-14.
2. Xu, F. F.; Imlay, J. A., Silver(I), mercury(II), cadmium(II), and zinc(II) target exposed enzymic iron-sulfur clusters when they toxify Escherichia coli. Applied and environmental microbiology 2012, 78 (10), 3614-3621.
3. (a) Harrison, J. J.; Tremaroli, V.; Stan, M. A.; Chan, C. S.; Vacchi-Suzzi, C.; Heyne, B. J.; Parsek, M. R.; Ceri, H.; Turner, R. J., Chromosomal antioxidant genes have metal ion-specific roles as determinants of bacterial metal tolerance. Environmental microbiology 2009, 11 (10), 2491-2509; (b) Lemire, J. A.; Harrison, J. J.; Turner, R. J., Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat Rev Micro 2013, 11 (6), 371-384.
4. Mermod, M.; Magnani, D.; Solioz, M.; Stoyanov, J. V., The copper-inducible ComR (YcfQ) repressor regulates expression of ComC (YcfR), which affects copper permeability of the outer membrane of Escherichia coli. BioMetals 2012, 25 (1), 33-43.
5. Björklöf, K.; Zickermann, V.; Finel, M., Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH-2) and analysis of its interaction with ubiquinone analogues. FEBS Letters 2000, 467 (1), 105-110.
6. Rapisarda, V. A.; Farı́, R. N.; Massa, E. M., Characterization of an NADH-Linked Cupric Reductase Activity from the Escherichia coli Respiratory Chain. Archives of Biochemistry and Biophysics 1999, 370 (2), 143-150.
7. Rapisarda, V. A.; Chehı́n, R. N.; De Las Rivas, J.; Rodrı́guez-Montelongo, L.; Farı́as, R. N.; Massa, E. M., Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2. Archives of Biochemistry and Biophysics 2002, 405 (1), 87-94.
8. Rodriguez-Montelongo, L.; Volentini, S. I.; Farias, R. N.; Massa, E. M.; Rapisarda, V. A., The Cu(II)-reductase NADH dehydrogenase-2 of Escherichia coli improves the bacterial growth in extreme copper concentrations and increases the resistance to the damage caused by copper and hydroperoxide. Archives of Biochemistry and Biophysics 2006, 451 (1), 1-7.
9. Outten, F. W.; Huffman, D. L.; Hale, J. A.; O'Halloran, T. V., The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli. The Journal of biological chemistry 2001, 276 (33), 30670-30677.
10. (a) Brown, N. L.; Stoyanov, J. V.; Kidd, S. P.; Hobman, J. L., The MerR family of transcriptional regulators. FEMS Microbiology Reviews 2003, 27 (2-3), 145-163; (b) Philips, S. J.; Canalizo-Hernandez, M.; Yildirim, I.; Schatz, G. C.; Mondragón, A.; O’Halloran, T. V., Allosteric transcriptional regulation via changes in the overall topology of the core promoter. Science 2015, 349 (6250), 877-881.
11. Rensing, C.; Fan, B.; Sharma, R.; Mitra, B.; Rosen, B. P., CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci U S A 2000, 97 (2), 652-656.
12. Djoko, K. Y.; Chong, L. X.; Wedd, A. G.; Xiao, Z., Reaction mechanisms of the multicopper oxidase CueO from Escherichia coli support its functional role as a cuprous oxidase. Journal of the American Chemical Society 2010, 132 (6), 2005-2015.
13. Gudipaty, S. A.; McEvoy, M. M., The histidine kinase CusS senses silver ions through direct binding by its sensor domain. Biochimica et biophysica acta 2014, 1844 (9), 1656-1661.
14. Rensing, C.; Grass, G., Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiology Reviews 2003, 27 (2-3), 197-213.
15. Kim, E. H.; Nies, D. H.; McEvoy, M. M.; Rensing, C., Switch or funnel: how RND-type transport systems control periplasmic metal homeostasis. Journal of bacteriology 2011, 193 (10), 2381-2387.
16. Chacon, K. N.; Mealman, T. D.; McEvoy, M. M.; Blackburn, N. J., Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. Proc Natl Acad Sci U S A 2014, 111 (43), 15373-15378.
17. Tetaz, T. J.; Luke, R. K., Plasmid-controlled resistance to copper in Escherichia coli. Journal of bacteriology 1983, 154 (3), 1263-1268.
18. Rouch, D. A.; Brown, N. L., Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco. Microbiology 1997, 143 (4), 1191-1202.
19. (a) Arnesano, F.; Banci, L.; Bertini, I.; Mangani, S.; Thompsett, A. R., A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites. Proc Natl Acad Sci U S A 2003, 100 (7), 3814-3819; (b) Djoko, K. Y.; Xiao, Z.; Huffman, D. L.; Wedd, A. G., Conserved mechanism of copper binding and transfer. A comparison of the copper-resistance proteins PcoC from Escherichia coli and CopC from Pseudomonas syringae. Inorganic chemistry 2007, 46 (11), 4560-4568.
20. Lee, S. M.; Grass, G.; Rensing, C.; Barrett, S. R.; Yates, C. J. D.; Stoyanov, J. V.; Brown, N. L., The Pco proteins are involved in periplasmic copper handling in Escherichia coli. Biochemical and Biophysical Research Communications 2002, 295 (3), 616-620.
21. Verma, N.; Singh, M., Biosensors for heavy metals. BioMetals 2005, 18 (2), 121-129.
22. Yagi, K., Applications of whole-cell bacterial sensors in biotechnology and environmental science. Applied microbiology and biotechnology 2007, 73 (6), 1251-1258.
23. Bereza-Malcolm, L. T.; Mann, G.; Franks, A. E., Environmental sensing of heavy metals through whole cell microbial biosensors: a synthetic biology approach. ACS synthetic biology 2015, 4 (5), 535-546.
24. Shemer, B.; Palevsky, N.; Yagur-Kroll, S.; Belkin, S., Genetically engineered microorganisms for the detection of explosives' residues. Frontiers in microbiology 2015, 6, 1175.
25. Changela, A.; Chen, K.; Xue, Y.; Holschen, J.; Outten, C. E.; O'Halloran, T. V.; Mondragon, A., Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003, 301 (5638), 1383-1387.
26. West, A. H.; Stock, A. M., Histidine kinases and response regulator proteins in two-component signaling systems. Trends in Biochemical Sciences 2001, 26 (6), 369-376.
27. Wang, S., Bacterial Two-Component Systems: Structures and Signaling Mechanisms. In Protein Phosphorylation in Human Health, Huang, C., Ed. InTech: 2012; p 439.
28. Gao, R.; Tao, Y.; Stock, A. M., System-level mapping of Escherichia coli response regulator dimerization with FRET hybrids. Molecular microbiology 2008, 69 (6), 1358-1372.
29. Bourret, R. B., Receiver domain structure and function in response regulator proteins. Current opinion in microbiology 2010, 13 (2), 142-149.
30. Galperin, M. Y., Structural classification of bacterial response regulators: diversity of output domains and domain combinations. Journal of bacteriology 2006, 188 (12), 4169-4182.
31. Singleton, C.; Hearnshaw, S.; Zhou, L.; Le Brun, N. E.; Hemmings, A. M., Mechanistic insights into Cu(I) cluster transfer between the chaperone CopZ and its cognate Cu(I)-transporting P-type ATPase, CopA. Biochem J 2009, 424 (3), 347-356.
32. Gonzalez-Guerrero, M.; Arguello, J. M., Mechanism of Cu+-transporting ATPases: soluble Cu+ chaperones directly transfer Cu+ to transmembrane transport sites. Proc Natl Acad Sci U S A 2008, 105 (16), 5992-5997.
33. Monchy, S.; Benotmane, M. A.; Wattiez, R.; van Aelst, S.; Auquier, V.; Borremans, B.; Mergeay, M.; Taghavi, S.; van der Lelie, D.; Vallaeys, T., Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34. Microbiology 2006, 152 (Pt 6), 1765-1776.
34. Sendra, V.; Cannella, D.; Bersch, B.; Fieschi, F.; Ménage, S.; Lascoux, D.; Covès, J., CopH from Cupriavidus metallidurans CH34. A Novel Periplasmic Copper-Binding Protein. Biochemistry 2006, 45 (17), 5557-5566.
35. Baird, G. S.; Zacharias, D. A.; Tsien, R. Y., Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 2000, 97 (22), 11984-11989.
36. Campbell, R. E.; Tour, O.; Palmer, A. E.; Steinbach, P. A.; Baird, G. S.; Zacharias, D. A.; Tsien, R. Y., A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 2002, 99 (12), 7877-7882.
37. Shaner, N. C.; Patterson, G. H.; Davidson, M. W., Advances in fluorescent protein technology. Journal of cell science 2007, 120 (Pt 24), 4247-4260.
38. (a) Sekiguchi, H.; Ozeki, Y.; Sasaki, N., In vitro synthesis of betaxanthins using recombinant DOPA 4,5-dioxygenase and evaluation of their radical-scavenging activities. Journal of agricultural and food chemistry 2010, 58 (23), 12504-12509; (b) Nakatsuka, T.; Yamada, E.; Takahashi, H.; Imamura, T.; Suzuki, M.; Ozeki, Y.; Tsujimura, I.; Saito, M.; Sakamoto, Y.; Sasaki, N.; Nishihara, M., Genetic engineering of yellow betalain pigments beyond the species barrier. Scientific reports 2013, 3, 1970.
39. Sasaki, N.; Abe, Y.; Goda, Y.; Adachi, T.; Kasahara, K.; Ozeki, Y., Detection of DOPA 4,5-dioxygenase (DOD) activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa. Plant & cell physiology 2009, 50 (5), 1012-1016.
40. Li, P. S.; Peng, Z. W.; Su, J.; Tao, H. C., Construction and optimization of a Pseudomonas putida whole-cell bioreporter for detection of bioavailable copper. Biotechnology letters 2014, 36 (4), 761-766.
41. Organization, W. H., Guidelines for drinking-water quality: recommendations. World Health Organization: 2004; Vol. 1.
42. Tseng, H. W.; Tsai, Y. J.; Yen, J. H.; Chen, P. H.; Yeh, Y. C., A fluorescence-based microbial sensor for the selective detection of gold. Chem Commun (Camb) 2014, 50 (14), 1735-1737.
43. Perez Audero, M. E.; Podoroska, B. M.; Ibanez, M. M.; Cauerhff, A.; Checa, S. K.; Soncini, F. C., Target transcription binding sites differentiate two groups of MerR-monovalent metal ion sensors. Molecular microbiology 2010, 78 (4), 853-865.
44. Vaara, M., Agents that increase the permeability of the outer membrane. Microbiological Reviews 1992, 56 (3), 395-411.
45. Yamamoto, K.; Ishihama, A., Transcriptional response of Escherichia coli to external copper. Molecular microbiology 2005, 56 (1), 215-227.
46. (a) Monchy, S.; Benotmane, M. A.; Janssen, P.; Vallaeys, T.; Taghavi, S.; van der Lelie, D.; Mergeay, M., Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans are specialized in the maximal viable response to heavy metals. Journal of bacteriology 2007, 189 (20), 7417-7425; (b) Reith, F.; Etschmann, B.; Grosse, C.; Moors, H.; Benotmane, M. A.; Monsieurs, P.; Grass, G.; Doonan, C.; Vogt, S.; Lai, B.; Martinez-Criado, G.; George, G. N.; Nies, D. H.; Mergeay, M.; Pring, A.; Southam, G.; Brugger, J., Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans. Proc Natl Acad Sci U S A 2009, 106 (42), 17757-17762.
47. Yeh, Y. C.; Muller, J.; Bi, C.; Hillson, N. J.; Beller, H. R.; Chhabra, S. R.; Singer, S. W., Functionalizing bacterial cell surfaces with a phage protein. Chem Commun (Camb) 2013, 49 (9), 910-912.
48. Ravikumar, S.; Pham, V. D.; Lee, S. H.; Yoo, I. K.; Hong, S. H., Modification of CusSR bacterial two-component systems by the introduction of an inducible positive feedback loop. Journal of industrial microbiology & biotechnology 2012, 39 (6), 861-868.
49. Cerminati, S.; Soncini, F. C.; Checa, S. K., Selective detection of gold using genetically engineered bacterial reporters. Biotechnology and bioengineering 2011, 108 (11), 2553-2560.