臺灣博碩士論文加值系統

生物感知器近年來常被用來替代常規檢測方法以監測公共衛生與生態系統中的 重金屬污染。然而，生物感知器對金屬靈敏度與專一性間相抵觸的權衡問題，相對 地阻礙了其發展。為解決此困境，本研究採用遺傳工程方法，發展出對鎘金屬具有 高靈敏度、專一性與反應性的生物感知器。我們在大腸桿菌細胞中植入帶有 CadR 轉 錄因子與其同源調控啟動子的基因模組，並透過剔除大腸桿菌的金屬外運蛋白來增 加細胞中鎘金屬的囤積量，從而加強了帶有 CadR 轉錄因子同源物的大腸桿菌其對鎘 金屬的靈敏度。另一方面，在改造的大腸桿菌中消去其他金屬的影響也使得最終的 生物感知器對鎘金屬具有高度專一性。此生物感知器可在 3 nM 的鎘金屬誘導下監測 到細胞螢光，並在 0-200 nM 間對鎘金屬具有線性的反應，且相對於無加金屬的狀態， 此生物感知器在鎘金屬誘導下能產生高達 777 倍的螢光信號變化。此外，我結合了此 生物感知器與智慧型手機開發出一套簡便的檢測方法，可用以偵測農業灌溉水與人 體尿液中的鎘金屬濃度。此方法具有高成本效益、易於使用與可擴展至篩選大量農 業與醫學樣品等優點。另一方面，我也進行了偏極螢光測定來探討 CadR 同源物在細 胞中對金屬反應差異的內在機理。總結而言，本研究強調了生物感知器的潛力與實 際應用價值，並對鎘生物感知器的發展作出貢獻。

Recently, whole-cell biosensors have become a favorable alternative to conventional chemical methods for monitoring heavy metal pollution in public health and ecosystems. However, the inherent trade-off between sensitivity and specificity has hindered their development over decades. Here, I generated a sensitive, specific, and high-response whole- cell biosensor for cadmium ions through genetic engineering of Escherichia coli.
Genetic modules harboring CadR homologs and the cognate promoters were introduced into E. coli for cadmium detection. Reconfiguring the metal transport system of E. coli enabled the enrichment of intracellular cadmium ions, thereby enhancing the sensitivity of E. coli bearing CadR homologs to detect cadmium ions. Also, depriving interfering metal species in the engineered E. coli allowed it to respond to cadmium ions specifically. The resulting cadmium biosensor exhibited a detection limit of 3 nM, a linear response range from 0 to 200 nM, and a maximal 777-fold signal change. In addition, a smartphone-assisted method capable of measuring cadmium ions in irrigation water and human urine was developed. This assay was cost-effective, user-friendly and scalable to screen large amounts of agricultural and medical samples. Moreover, fluorescence polarization measurement was performed to dissect the molecular basis of metal reactivity of two CadR homologs in vitro. Collectively, my thesis work contributes to the develpoment of a cadmium biosensor and underscores the potential and value of whole-cell biosensors.

口試委員審定書 i
誌謝 ii
摘要 iii
Abstract iv
Contents v
Figures vi
Tables vii
Chapter I. Introduction 1
Chapter II. Development of a sensitive and specific whole-cell cadmium biosensor 7
Chapter III. Developing a smartphone-based cell pellet assay for cadmium quantification 31
Chapter IV. Investigating the binding mechanism of CadR homologs and DNA 38
Chapter V. Conclusions and future perspectives 53
Supplementaty information 55

Chapter I.
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Chapter II.
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15. Tang, X.; Zeng, G. M.; Fan, C. Z.; Zhou, M.; Tang, L.; Zhu, J. J.; Wan, J.; Huang, D. L.; Chen, M.; Xu, P.; Zhang, C.; Lu, Y.; Xiong, W. P., Chromosomal expression of CadR on Pseudomonas aeruginosa for the removal of Cd(II) from aqueous solutions. Sci Total Environ 2018, 636, 1355-1361.
16. Permina, E. A.; Kazakov, A. E.; Kalinina, O. V.; Gelfand, M. S., Comparative genomics of regulation of heavy metal resistance in Eubacteria. BMC Microbiol 2006, 6, 49.
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Chapter III.
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3. He, M. Y.; Lin, Y. J.; Kao, Y. L.; Kuo, P.; Grauffel, C.; Lim, C.; Cheng, Y. S.; Chou, H. D., Sensitive and Specific Cadmium Biosensor Developed by Reconfiguring Metal Transport and Leveraging Natural Gene Repositories. ACS Sens 2021.
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7. Zhang, L. F.; Hu, W. P.; Yu, L. P.; Wang, Y., Click synthesis of a novel triazole bridged AIE active cyclodextrin probe for specific detection of Cd-2. Chem Commun 2015, 51 (20), 4298- 4301.
8. Lin, L.; Wang, Y.; Xiao, Y.; Liu, W., Hydrothermal synthesis of carbon dots codoped with nitrogen and phosphorus as a turn-on fluorescent probe for cadmium(II). Mikrochim Acta 2019, 186 (3), 147.
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Chapter IV.
1. Lee, S. W.; Glickmann, E.; Cooksey, D. A., Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator. Appl Environ Microb 2001, 67 (4), 1437-1444.
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6. Liu, X.; Hu, Q.; Yang, J.; Huang, S.; Wei, T.; Chen, W.; He, Y.; Wang, D.; Liu, Z.; Wang, K.; Gan, J.; Chen, H., Selective cadmium regulation mediated by a cooperative binding mechanism in CadR. Proc Natl Acad Sci U S A 2019, 116 (41), 20398-20403.
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9. Joshi, C. P.; Panda, D.; Martell, D. J.; Andoy, N. M.; Chen, T. Y.; Gaballa, A.; Helmann, J. D.; Chen, P., Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator. Proc Natl Acad Sci U S A 2012, 109 (38), 15121-6.
10. Andoy, N. M.; Sarkar, S. K.; Wang, Q.; Panda, D.; Benitez, J. J.; Kalininskiy, A.; Chen, P., Single-molecule study of metalloregulator CueR-DNA interactions using engineered Holliday junctions. Biophys J 2009, 97 (3), 844-52.
11. Osman, D.; Foster, A. W.; Chen, J. J.; Svedaite, K.; Steed, J. W.; Lurie-Luke, E.; Huggins, T. G.; Robinson, N. J., Fine control of metal concentrations is necessary for cells to discern zinc from cobalt. Nature Communications 2017, 8.
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Chapter V.
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Supplementary information
1. He, M. Y.; Lin, Y. J.; Kao, Y. L.; Kuo, P.; Grauffel, C.; Lim, C.; Cheng, Y. S.; Chou, H. D., Sensitive and Specific Cadmium Biosensor Developed by Reconfiguring Metal Transport and Leveraging Natural Gene Repositories. ACS Sens 2021.