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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊啓伸 | zh_TW |
dc.contributor.advisor | Chii-Shen Yang | en |
dc.contributor.author | 游承翰 | zh_TW |
dc.contributor.author | Cheng-Han Yu | en |
dc.date.accessioned | 2023-08-09T16:14:39Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-08-09 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-07-20 | - |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88257 | - |
dc.description.abstract | 微生物視紫質 (microbial rhodopsin) 為一類由七個穿膜螺旋 (transmembrane helix) 及全反式視黃醛發色團 (all-trans-retinal chromophore) 所組成的感光膜蛋白質。在自然界中,微生物視紫質參與多項生理功能,如離子運輸及趨光性 (phototaxis)。此類蛋白質在特定波長的光刺激下行使其功能,並產生一系列稱為光週期 (photocycle) 的構形改變 (conformational change)。因此,對離子幫浦型微生物視紫質而言,光週期的反應動態與離子選擇性便成為其運輸活性的決定因子。在嗜鹽古菌 (halophilic archaea) 中,有兩種廣受關注的離子幫浦型微生物視紫質,即菌視紫質 (bacteriorhodopsin, BR) 與氯視紫質 (halorhodopsin, HR)。菌視紫質與氯視紫質分別為氫離子及氯離子幫浦,其功能皆會導致細胞過極化 (hyperpolarization),該特性也促成兩者於光控基因生物學上的應用。為探究微生物視紫質的離子運輸對細胞內電位之影響,我們將菌視紫質與氯視紫質表現於人類胚胎腎細胞株 HEK293 及大腸桿菌 (Escherichia coli) 中,以建立適用於兩表現系統的膜片箝制記錄 (patch-clamp recording) 裝置。相較於 HEK293 細胞株的廣泛應用,大腸桿菌須透過巨型原膜體 (giant spheroplast) 的製備使其見用於電生理研究。巨型原膜體乃透過抑制細胞分裂及移除細胞壁來產生。本研究選用 Haloarcula marismortui BRI 的 D94N 突變型(亦稱 highly expressible BR, HEBR)與 Natronomonas pharaonis 的氯視紫質 (NpHR) 作為研究光週期與離子運輸相關性之蛋白質標的。我們發現 HEBR 的光週期時間常數約為其野生型的 50 倍長,而 NpHR 者則與前人實驗相符。在 HEK293 細胞實驗中,HEBR、NpHR 兩者皆成功進行轉染。後續的全細胞電壓箝制實驗 (whole-cell voltage-clamp recording) 則能在負細胞電位下記錄到 HEBR 轉染株的光電流 (photocurrent)。大腸桿菌的膜片箝制實驗方面,本研究提出以下有助於製備表現菌視紫質與氯視紫質之巨型原膜體的步驟:1)誘導蛋白質表現後,不進行過夜 2°C 培養、2)以置換緩衝溶液的方式終止細胞壁裂解反應、3)以 2°C 進行保存。在確認 NpHR 於大腸桿菌巨型原膜體中的功能性後,我們正致力於完成細胞膜封接 (membrane seal) 以在大腸桿菌中測量光電流。本研究旨在以膜片箝制實驗研究微生物視紫質光週期對離子運輸的影響,並以此分析方法建立一適用真核及原核表現系統之光控基因生物學工具篩選平台。 | zh_TW |
dc.description.abstract | Microbial rhodopsins (MRhos) are a family of light-sensitive membrane proteins composed of seven transmembrane helices and an embedded all-trans-retinal (ATR) chromophore. These proteins exert diverse functions, participating in ion transport and phototaxis. Upon light stimulation of specific wavelengths, mRhos function while undergoing a series of conformational changes termed photocycle. Photocycle kinetics and ion selectivity thus determine the transport activity of ion-pumping mRhos. Bacteriorhodopsin (BR) and halorhodopsin (HR) are two well-studied mRhos from halophilic archaea. BR and HR are a proton and a chloride pump, respectively. Both mRhos’ functionality leads to cell hyperpolarization, inspiring their optogenetic applications in neuroscience. To investigate how ion transport of mRhos affects the whole-cell potential, we expressed the proteins in HEK293 and Escherichia coli cells. We aimed to construct a patch-clamp device applicable to both expression systems. HEK293 is a cell line readily amenable to patch-clamp measurements. On the other hand, previous studies applied E. coli cells in patch-clamp recordings by preparing giant spheroplasts via inhibition of cell division and removal of the cell wall. We selected the D94N mutant of Haloarcula marismortui BRI (a highly expressible BR, HEBR) and the HR from Natronomonas pharaonis (NpHR) to investigate the effects of photocycle on ion transport. We found that the photocycle time constant of HEBR is about 50-fold longer than its wild type. The photocycle kinetics of our NpHR construct is comparable to previous studies. BR and HR constructs were successfully transfected into HEK293 cells. Subsequent whole-cell voltage-clamp recordings displayed the photocurrents of HEBR transfectants under membrane voltages of -60 to 20 mV. For patch-clamp experiments on E. coli giant spheroplasts, we identified favorable preparation procedures for BR- and HR-expressing cells: 1) skipping overnight refrigeration after induction, 2) terminating the cell wall digestion by buffer replacement, and 3) storing at 2°C. The functionality of NpHR was confirmed in giant spheroplasts, and we aimed to attain photocurrents after forming the membrane seal. Through further analyzing the correlation between the photocycle kinetics and ion transport of mRhos, our patch-clamp system has the potential to be a handy screening platform for optogenetic tools using HEK293 and E. coli cells. | en |
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dc.description.tableofcontents | Acknowledgements i
摘要 ii Abstract iv Table of Contents vi List of Figures x List of Tables xii List of Abbreviations xiii Chapter 1 Introduction 1 1.1 Microbial rhodopsin 1 1.1.1 Photochemical properties of microbial rhodopsin 1 1.1.2 Functionality and physiology of microbial rhodopsin 3 1.1.3 Bacteriorhodopsin 4 1.1.4 Halorhodopsin 5 1.2 Optogenetic applications of microbial rhodopsin 7 1.3 Patch-clamp electrophysiology 8 1.3.1 Patch-clamp recording techniques 8 1.3.2 Patch-clamp studies of microbial rhodopsin 10 1.4 Giant spheroplasts 11 1.4.1 Bacterial spheroplasts and giant spheroplasts 11 1.4.2 E. coli giant spheroplasts in the electrophysiological research 12 1.5 Purpose of this study 13 Chapter 2 Materials and Methods 15 2.1 Materials 15 2.1.1 Bacterial strains 15 2.1.2 Cell lines 15 2.1.3 Plasmid constructs 15 2.1.4 Chemical reagents 16 2.2 Apparatus and Software 18 2.2.1 Cell cultivation 18 2.2.2 Centrifugation 18 2.2.3 Laser flash photolysis system 19 2.2.4 LV-ITO-based photocurrent measurement system 19 2.2.5 Microscopy 20 2.2.6 Patch-clamp system 20 2.2.7 Software 21 2.2.8 Miscellaneous 21 2.3 Methods 22 2.3.1 Protein expression and purification 22 2.3.2 UV-vis spectroscopy 24 2.3.3 Laser flash photolysis 24 2.3.4 HEK293 cell cultivation and transfection 25 2.3.5 Microscopy and image processing for HEK293 cells 26 2.3.6 Preparation of E. coli spheroplasts and giant spheroplasts 27 2.3.7 LV-ITO-based photocurrent measurement 29 2.3.8 Patch-clamp recording 30 Chapter 3 Results 34 3.1 Functionality of HEBR and NpHR proteins 34 3.2 Expression of mRhos in HEK293 cells 36 3.2.1 Transfection efficiency of mRho-EYFP constructs in HEK293 cells 36 3.2.2. Localization of mRho-EYFP proteins in HEK293 cells 39 3.3 Electrophysiology of mRhos expressed in HEK293 cells 42 3.4 Expression of mRhos in E. coli giant spheroplasts 45 3.4.1 Protocol modifications for preparing E. coli giant spheroplasts 45 3.4.2 Light-driven ion transport of NpHR in E. coli spheroplasts and giant spheroplasts 47 Chapter 4 Discussion and Prospects 49 4.1 Expression and patch-clamp recording of mRhos in HEK293 cells 49 4.2 Photocurrents of HEBR expressed in HEK293 cells 51 4.3 Patch-clamp attempts on E. coli giant spheroplasts 52 4.4 Potential applications 55 References 57 Supplementary Information 67 Supplementary methods 67 1. The ImageJ macro script implemented in this study 67 Supplementary figures 70 | - |
dc.language.iso | en | - |
dc.title | 測定微生物視紫質之膜片箝制系統的建立 | zh_TW |
dc.title | Construction of patch-clamp systems for characterizing microbial rhodopsins | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.coadvisor | 櫻井啓輔 | zh_TW |
dc.contributor.coadvisor | Keisuke Sakurai | en |
dc.contributor.oralexamcommittee | 傅煦媛;林宥成 | zh_TW |
dc.contributor.oralexamcommittee | Hsu-Yuan Fu;Yu-Cheng Lin | en |
dc.subject.keyword | 微生物視紫質,HEK293,巨型原膜體,膜片箝制記錄術,光電流,光控基因生物學, | zh_TW |
dc.subject.keyword | Microbial rhodopsin,HEK293,Giant spheroplast,Patch-clamp recording,Photocurrent,Optogenetics, | en |
dc.relation.page | 73 | - |
dc.identifier.doi | 10.6342/NTU202301303 | - |
dc.rights.note | 同意授權(限校園內公開) | - |
dc.date.accepted | 2023-07-20 | - |
dc.contributor.author-college | 生命科學院 | - |
dc.contributor.author-dept | 生化科技學系 | - |
顯示於系所單位: | 生化科技學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-111-2.pdf 目前未授權公開取用 | 3.5 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。