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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周信宏 | |
| dc.contributor.author | Pu Kuo | en |
| dc.contributor.author | 郭璞 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:28:23Z | - |
| dc.date.copyright | 2020-07-15 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-06-03 | |
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PLoS ONE 8 (8): e69292. doi:10.1371/journal.pone.0069292. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15188 | - |
| dc.description.abstract | 生物必須維持體內金屬離子的恆定。在大腸桿菌中，為保持細胞質內的單價銅離子恆定，一種名為CueR的MerR蛋白家族轉錄因子為關鍵之一。CueR 對單價銅離子有高親和性，並且與銀離子和金離子皆會結合。透過隨機突變CueR金屬結合域的胺基酸， 前人研究發現S117P及D116P 突變型皆相較野生型對金離子更加具有選擇性。我利用凝膠電泳遷移率實驗及等溫滴定量熱法，剖析影響S117P及D116P的金屬選擇性之生化機制，並發現金屬離子誘發的啟動子DNA構形改變是造成S117P對金離子有高選擇性的主要因子。除了研究CueR突變種的生化特性以外，我嘗試以CueR-S117P以及大腸桿菌改造株製作成全細胞的生物感測器。此生物感測器對金離子有優秀的選擇性，並且在金離子濃度為0.1至2 µM之間有線性的反應曲線。總結而言，本研究增進了學界對蛋白質金屬選擇性的理解，並且為金屬生物感測器的發展作出貢獻。 | zh_TW |
| dc.description.abstract | Maintaining the homeostasis of metal ions is crucial for living organisms. In Escherichia coli, a MerR family transcription factor named CueR is critical for copper homeostasis in the cytosol. CueR has high affinity to monovalent copper and binds to silver or gold ions as well. Previous mutagenesis of the CueR metal-binding domain demonstrated that mutations S117P and D116P were more selective to gold than the wild-type (WT). I aimed to dissect the biochemical mechanisms underlying the gold specificity of S117P and D116P by the electrophoretic mobility shift assay and isothermal titration calorimetry. I found that the relative conformational change of PcopA, a cueR-regulated promoter, correlated with the level of transcriptional activation by CueR-WT and the two mutants. In addition to studying the biochemical properties of CueR mutants, I used the CueR-S117P and an E. coli strain with compromised metal homeostasis to develop a whole-cell biosensor for gold cations. This biosensor had high selectivity for gold and a linear response range between 0.1 and 2 µM. Collectively, my work provided mechanistic insights into the cation selectivity of a metalloprotein and contributed to the development of a gold biosensor. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:28:23Z (GMT). No. of bitstreams: 1 ntu-109-R06b21020-1.pdf: 4147331 bytes, checksum: 745e2c30f3e1a656dc61ade4f9689038 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 摘要 iii Abstract iv Contents v Figures vi Tables viii List of abbreviations ix Chapter I. Introduction 1 Chapter II. Biochemical characterization of gold-specific CueR mutants 7 Summary 7 Introduction 7 Materials and methods 9 Results 13 2.1. Expression and purification of CueR 13 2.2 Binding affinity of CueR and PcopA 15 2.3 Measuring the affinity of CueR protein and metal cations by isothermal titration calorimetry 20 2.4 Metal-induced conformational change of the CueR-PcopA complex 25 Discussion 30 References 33 Chapter III. CueR-S117P as a potential biosensor 38 Introduction 38 Materials and Methods 39 Results 41 Discussion 43 References 45 Chapter IV. Conclusion & Future Perspectives 48 | |
| dc.language.iso | en | |
| dc.subject | 金屬蛋白 | zh_TW |
| dc.subject | 凝膠電泳遷移率實驗 | zh_TW |
| dc.subject | 等溫滴定量熱法 | zh_TW |
| dc.subject | CueR | zh_TW |
| dc.subject | MerR蛋白質家族 | zh_TW |
| dc.subject | 生物感測器 | zh_TW |
| dc.subject | biosensor | en |
| dc.subject | CueR | en |
| dc.subject | Metalloprotein | en |
| dc.subject | EMSA | en |
| dc.subject | ITC | en |
| dc.subject | MerR family | en |
| dc.title | 金選擇性生物感測器之特徵描述及發展 | zh_TW |
| dc.title | Characterization and development of gold-selective biosensors | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭貽生,葉怡均 | |
| dc.subject.keyword | 金屬蛋白,凝膠電泳遷移率實驗,等溫滴定量熱法,CueR,MerR蛋白質家族,生物感測器, | zh_TW |
| dc.subject.keyword | Metalloprotein,EMSA,ITC,CueR,MerR family,biosensor, | en |
| dc.relation.page | 49 | |
| dc.identifier.doi | 10.6342/NTU202000935 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-06-04 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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