我們實驗室提出以全細胞生物感測器來有效檢測並定量水中的硝酸鹽及亞硝酸鹽的方法，參考葡萄糖在大腸桿菌中的代謝路徑建構之質體轉殖入 E. coli BL21(DE3)，其製造的T7 RNA polymerse (RNAP)會開啟T7啟動子製造下游蛋白，這些蛋白會加速葡萄糖代謝成為4-Aminobenzoic acid (PABA)，使我們在短時間內就可以得到大量PABA，其經由一系列反應得到最終產物在540 nm有最大吸收的色素，以吸收回推濃度、再以大腸桿菌還原硝酸鹽為亞硝酸鹽後並以同樣反應定量出硝酸鹽濃度。

In this study, we propose a method for the efficient detection and quantification of nitrate and nitrite in water using biosynthesis product 4-Aminobenzoic acid (PABA). Referring to the metabolic pathway of glucose in E. coli, the plasmid constructed is transformed into E.coli BL21(DE3). The T7 RNA polymerse (RNAP) produced by E. coli BL21(DE3) will turn on the T7 promoter to produce downstream proteins, which will accelerate glucose metabolism into PABA, so that we can obtain a large amount of PABA in a short time. PABA passes through a series of reactions to obtained the pigment as final product, which the maximum absorbance is at 540 nm. The concentration is calculated by the absorbance, and the nitrate is reduced by E. coli and quantified by the same reaction.

Ziegler, C.; Göpel, W., Biosensor development. Current opinion in chemical biology 1998, 2 (5), 585-591.
Gu, M. B.; Mitchell, R. J.; Kim, B. C., Whole-cell-based biosensors for environmental biomonitoring and application. Biomanufacturing 2004, 269-305.
Tenaillon, O.; Skurnik, D.; Picard, B.; Denamur, E., The population genetics of commensal Escherichia coli. Nature reviews microbiology 2010, 8 (3), 207-217.
Nataro, J. P.; Kaper, J. B., Diarrheagenic escherichia coli. Clinical microbiology reviews 1998, 11 (1), 142-201.
Erlich, H. A.; Gelfand, D.; Sninsky, J. J., Recent advances in the polymerase chain reaction. Science 1991, 252 (5013), 1643-1651.
Anton, B. P.; Raleigh, E. A., Complete genome sequence of NEB 5-alpha, a derivative of Escherichia coli K-12 DH5α. Genome announcements 2016, 4 (6), e01245-16.
Jeong, H.; Barbe, V.; Lee, C. H.; Vallenet, D.; Yu, D. S.; Choi, S.-H.; Couloux, A.; Lee, S.-W.; Yoon, S. H.; Cattolico, L., Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3). Journal of molecular biology 2009, 394 (4), 644-652.
Dumon-Seignovert, L.; Cariot, G.; Vuillard, L., The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21 (DE3), C41 (DE3), and C43 (DE3). Protein expression purification 2004, 37 (1), 203-206.
Holms, W., The central metabolic pathways of Escherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. Current topics in cellular regulation 1986, 28, 69-105.
Baron, S., Medical microbiology. 1996.
Kogure, T.; Inui, M., Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products. Applied microbiology biotechnology 2018, 102, 8685-8705.
Nam, T.-W.; Cho, S.-H.; Shin, D.; Kim, J.-H.; Jeong, J.-Y.; Lee, J.-H.; Roe, J.-H.; Peterkofsky, A.; Kang, S.-O.; Ryu, S., The Escherichia coli glucose transporter enzyme IICBGlc recruits the global repressor Mlc. The EMBO journal 2001, 20 (3), 491-498.
Postma, P. W.; Lengeler, J. W.; Jacobson, G., Phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria. Microbiological reviews 1993, 57 (3), 543-594.
Escalante, A.; Salinas Cervantes, A.; Gosset, G.; Bolívar, F., Current knowledge of the Escherichia coli phosphoenolpyruvate–carbohydrate phosphotransferase system: peculiarities of regulation and impact on growth and product formation. Applied microbiology biotechnology 2012, 94, 1483-1494.
Stincone, A.; Prigione, A.; Cramer, T.; Wamelink, M. M.; Campbell, K.; Cheung, E.; Olin‐Sandoval, V.; Grüning, N. M.; Krüger, A.; Tauqeer Alam, M., The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biological Reviews 2015, 90 (3), 927-963.
Gupta, R.; Gupta, N., Fundamentals of Bacterial Physiology and Metabolism. Springer: 2021.
Atwell, B. J.; Kriedemann, P. E.; Turnbull, C. G., Plants in action: adaptation in nature, performance in cultivation. Macmillan Education AU: 1999.
Herrmann, K. M.; Weaver, L. M., The shikimate pathway. Annual review of plant biology 1999, 50 (1), 473-503.
Nunes, J. E.; Duque, M. A.; de Freitas, T. F.; Galina, L.; Timmers, L. F.; Bizarro, C. V.; Machado, P.; Basso, L. A.; Ducati, R. G., Mycobacterium tuberculosis shikimate pathway enzymes as targets for the rational design of anti-tuberculosis drugs. Molecules 2020, 25 (6), 1259.
Bender, S. L.; Mehdi, S.; Knowles, J. R., Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis. Biochemistry 1989, 28 (19), 7555-7560.
Santos-Sánchez, N. F.; Salas-Coronado, R.; Hernández-Carlos, B.; Villanueva-Cañongo, C., Shikimic acid pathway in biosynthesis of phenolic compounds. Plant physiological aspects of phenolic compounds 2019, 1, 1-15.
Green, J. M.; Matthews, R. G., Folate biosynthesis, reduction, and polyglutamylation and the interconversion of folate derivatives. EcoSal Plus 2007, 2 (2).
Maynard, C.; Cummins, I.; Green, J.; Weinkove, D., Folic acid instability and bacterial metabolism combine to impact C. elegans development and ageing.
Maynard, C.; Weinkove, D., Bacteria increase host micronutrient availability: mechanisms revealed by studies in C. elegans. Genes nutrition 2020, 15 (1), 1-11.
Koma, D.; Yamanaka, H.; Moriyoshi, K.; Sakai, K.; Masuda, T.; Sato, Y.; Toida, K.; Ohmoto, T., Production of p-aminobenzoic acid by metabolically engineered Escherichia coli. Bioscience, Biotechnology, Biochemistry 2014, 78 (2), 350-357.
Kubota, T.; Watanabe, A.; Suda, M.; Kogure, T.; Hiraga, K.; Inui, M., Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct. Metabolic engineering 2016, 38, 322-330.
Griess test. https://en.wikipedia.org/wiki/Griess_test
Salama, M. F.; Abbas, A.; Darweish, M. M.; El-Hawwary, A. A.; Al-Gayyar, M. M., Hepatoprotective effects of cod liver oil against sodium nitrite toxicity in rats. Pharmaceutical biology 2013, 51 (11), 1435-1443.
Fan, A. M.; Steinberg, V. E., Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Regulatory toxicology pharmacology 1996, 23 (1), 35-43.
Walker, R., Nitrates, nitrites and N‐nitrosocompounds: A Review of the Occurrence in Food and Diet and the Toxicological Implications. Food Additives Contaminants 1990, 7 (6), 717-768.
Kyrtopoulos, S., N-nitroso compound formation in human gastric juice. Cancer surveys 1989, 8 (2), 423-442.
EPA, U., United States Environmental Protection Agency, National primary drinking water regulations: In Radionuclides Rule. Federal Register 2001, 76708-76753.
MacArthur, P. H.; Shiva, S.; Gladwin, M. T., Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence. Journal of Chromatography B 2007, 851 (1-2), 93-105.
Lin, Z.; Xue, W.; Chen, H.; Lin, J.-M., Peroxynitrous-acid-induced chemiluminescence of fluorescent carbon dots for nitrite sensing. Analytical chemistry 2011, 83 (21), 8245-8251.
Lin, Z.; Dou, X.; Li, H.; Ma, Y.; Lin, J.-M., Nitrite sensing based on the carbon dots-enhanced chemiluminescence from peroxynitrous acid and carbonate. Talanta 2015, 132, 457-462.
Sui, Y.; Deng, M.; Xu, S.; Chen, F., Gold nanocluster-enhanced peroxynitrous acid chemiluminescence for high selectivity sensing of nitrite. RSC advances 2015, 5 (18), 13495-13501.
Gill, A.; Zajda, J.; Meyerhoff, M. E., Comparison of electrochemical nitric oxide detection methods with chemiluminescence for measuring nitrite concentration in food samples. Analytica chimica acta 2019, 1077, 167-173.
Li, H.; Meininger, C. J.; Wu, G., Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B: Biomedical Sciences Applications 2000, 746 (2), 199-207.
Butt, S. B.; Riaz, M.; Iqbal, M. Z., Simultaneous determination of nitrite and nitrate by normal phase ion-pair liquid chromatography. Talanta 2001, 55 (4), 789-797.
Jobgen, W. S.; Jobgen, S. C.; Li, H.; Meininger, C. J.; Wu, G., Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography. Journal of Chromatography B 2007, 851 (1-2), 71-82.
Campanella, B.; Onor, M.; Pagliano, E., Rapid determination of nitrate in vegetables by gas chromatography mass spectrometry. Analytica Chimica Acta 2017, 980, 33-40.
Murray, E.; Roche, P.; Briet, M.; Moore, B.; Morrin, A.; Diamond, D.; Paull, B., Fully automated, low-cost ion chromatography system for in-situ analysis of nitrite and nitrate in natural waters. Talanta 2020, 216, 120955.
Melanson, J. E.; Lucy, C. A., Ultra-rapid analysis of nitrate and nitrite by capillary electrophoresis. Journal of Chromatography A 2000, 884 (1-2), 311-316.
Öztekin, N.; Nutku, M. S.; Erim, F. B., Simultaneous determination of nitrite and nitrate in meat products and vegetables by capillary electrophoresis. Food chemistry 2002, 76 (1), 103-106.
Della Betta, F.; Vitali, L.; Fett, R.; Costa, A. C. O., Development and validation of a sub-minute capillary zone electrophoresis method for determination of nitrate and nitrite in baby foods. Talanta 2014, 122, 23-29.
Kalaycıoğlu, Z.; Erim, F. B., Simultaneous determination of nitrate and nitrite in fish products with improved sensitivity by sample stacking-capillary electrophoresis. Food analytical methods 2016, 9, 706-711.
Kamilova, N.; Kalaycıoğlu, Z.; Gölcü, A. e. l., Sample Stacking–Capillary Electrophoretic Analysis of Nitrate and Nitrite in Organic-and Conventional-Originated Baby Food Formulas from Turkey. ACS Omega 2023.
Kazemzadeh, A.; Ensafi, A. A., Simultaneous determination of nitrite and nitrate in various samples using flow-injection spectrophotometric detection. Microchemical Journal 2001, 69 (2), 61-68.
Miranda, K. M.; Espey, M. G.; Wink, D. A., A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric oxide 2001, 5 (1), 62-71.
Cheng, Y.-H.; Kung, C.-W.; Chou, L.-Y.; Vittal, R.; Ho, K.-C., Poly (3, 4-ethylenedioxythiophene)(PEDOT) hollow microflowers and their application for nitrite sensing. Sensors Actuators B: Chemical 2014, 192, 762-768.
García-Robledo, E.; Corzo, A.; Papaspyrou, S., A fast and direct spectrophotometric method for the sequential determination of nitrate and nitrite at low concentrations in small volumes. Marine Chemistry 2014, 162, 30-36.
Zhang, L.; Wu, X.; Yuan, Z.; Lu, C., π-Conjugated thiolate amplified spectrophotometry nitrite assay with improved sensitivity and accuracy. Chemical Communications 2018, 54 (86), 12178-12181.
Radhakrishnan, S.; Krishnamoorthy, K.; Sekar, C.; Wilson, J.; Kim, S. J., A highly sensitive electrochemical sensor for nitrite detection based on Fe2O3 nanoparticles decorated reduced graphene oxide nanosheets. Applied Catalysis B: Environmental 2014, 148, 22-28.
Kung, C.-W.; Chang, T.-H.; Chou, L.-Y.; Hupp, J. T.; Farha, O. K.; Ho, K.-C., Porphyrin-based metal–organic framework thin films for electrochemical nitrite detection. Electrochemistry Communications 2015, 58, 51-56.
Haldorai, Y.; Kim, J. Y.; Vilian, A. E.; Heo, N. S.; Huh, Y. S.; Han, Y.-K., An enzyme-free electrochemical sensor based on reduced graphene oxide/Co3O4 nanospindle composite for sensitive detection of nitrite. Sensors Actuators B: Chemical 2016, 227, 92-99.
Li, X.; Ping, J.; Ying, Y., Recent developments in carbon nanomaterial-enabled electrochemical sensors for nitrite detection. TrAC Trends in Analytical Chemistry 2019, 113, 1-12.
Nasraoui, S.; Al-Hamry, A.; Teixeira, P. R.; Ameur, S.; Paterno, L. G.; Ali, M. B.; Kanoun, O., Electrochemical sensor for nitrite detection in water samples using flexible laser-induced graphene electrodes functionalized by CNT decorated by Au nanoparticles. Journal of Electroanalytical Chemistry 2021, 880, 114893.
Restriction Endonucleases: Molecular Cloning and Beyond. https://international.neb.com/products/restriction-endonucleases/restriction-endonucleases/restriction-endonucleases-molecular-cloning-and-beyond.
Golden Gate Assembly. https://international.neb.com/golden-gate/golden-gate.
KLD Enzyme Mix. https://international.neb.com/products/m0554-kld-enzyme-mix#Product%20Information.
Aukema, K. G.; Wackett, L. P., Inexpensive microbial dipstick diagnostic for nitrate in water. Environmental Science: Water Research Technology 2019, 5 (2), 406-416.
Váradi, L.; Breedon, M.; Chen, F. F.; Trinchi, A.; Cole, I. S.; Wei, G., Evaluation of novel Griess-reagent candidates for nitrite sensing in aqueous media identified via molecular fingerprint searching. RSC advances 2019, 9 (7), 3994-4000.
Wang, H.; Gunsalus, R. P., Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate, nitrite, and formate: roles for NarL and NarP. Journal of bacteriology 2003, 185 (17), 5076-5085.
Kiley, P. J.; Beinert, H., Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster. FEMS microbiology reviews 1998, 22 (5), 341-352.
Pinske, C.; Bönn, M.; Krüger, S.; Lindenstrauß, U.; Sawers, R. G., Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21 (DE3). PLoS One 2011, 6 (8), e22830.