Probing Escherichia coli Energetics under Starvation by Single-Cell Measurements

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林思遠(Ssu-Yuan Lin) 查詢紙本館藏

畢業系所

物理學系

論文名稱

(Probing Escherichia coli Energetics under Starvation by Single-Cell Measurements)

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摘要(中)

細菌需要多少能量生存?
細菌的存活是否存在最低能量需求?

能量對於生命來說十分重要，生物體內的生合成反應無一不需要能量的交換。然而又該如何定義生命呢? 我們可以將生命看做是一個過程，藉由基因儲存的資訊將環境中的物質製造成生命組成所需。然而現今生物學者大部分的努力都放在了解基因資訊上，而對能量的流動、利用途徑了解的很少。我們從超級細菌的危機意識到更基礎的了解是必要的，一昧的改變抗生素結構只會產生更多擁有多重抗藥性的超級細菌，我們試著從不一樣的角度切入解決，從基本的能量角度去了解細菌如何反應環境的變化。因此，我們嘗試用單細胞量測實驗來了解細菌在極端的環境，長時間處於飢餓狀態下體內的能量變化。
目前我們知道細菌體內有兩大主要的能量狀態，adenosine triphosphate (ATP)以及proton motive force (PMF)。ATP是在各種生命形式內普遍存在的能量貨幣，而PMF則是電位能和化學能共同貢獻而成，讓質子從細胞膜外進入膜內的位能。在這個論文研究中，我們利用螢光蛋白以及細菌鞭毛馬達來量測ATP以及PMF，而這個螢光蛋白是一種對ATP數量有反應的綠色螢光蛋白，細菌鞭毛馬達則是藉由質子流來驅動。從這兩種新穎的生物物理量測方式，我們可以從單細胞量測ATP以及PMF這兩種主要能量狀態。
我們的目標是希望藉由在長時間飢餓的環境下，得到單隻細菌可以利用的能量有多少，以及是否有個細菌生存最低能量需求的閾值，讓我們可以區別細菌的生與死。在我們將細菌換到完全沒有養分的環境下，細菌鞭毛馬達轉動的量測得到馬達在不同負載下轉速有不同的下降率，意味著馬達隨負載不同而有不同輸出功率。從我們長時間飢餓環境的實驗結果可以得到細菌消耗掉的能量約10-13焦耳，且與負載無關。接著我們在細菌鞭毛馬達停止轉動後，也就是沒有PMF後，將細菌重新換回有養分的環境，驚人的是所有的細菌幾乎都能夠在一天內恢復正常狀態並且生長分裂。而我們利用螢光蛋白量測細菌體內ATP濃度的實驗同樣得到與細菌鞭毛馬達量測的結果。這些單細菌量測實驗讓我們有機會對細菌體內能量的即時變化有更進一步的可能。

摘要(英)

‘’How much energy does a bacteria cell require to survive?’’
‘’Is there a minimum energy requirement for a bacterial cell to maintain viability?’’

Energy is crucial to life. A simplistic view of life is to think of it as a process that transforms external materials into cellular components based on genetic information and by transducing energy. Energy is required for all of the biological processes. While most of the efforts by biologists temp to understand the flow of genetic information, very little is known regarding cellular energetic flow. Furthermore, the crisis of superbugs, inspire us to focus on the fundamental understanding of bacterial energetic for alternative route of antibiotic discovery. Therefore, we conduct single-cell energetic experiments to probe the dynamics and boundary between life and death of bacteria under extreme starvation.
There are two major types of energy sources in bacteria, adenosine triphosphate (ATP) and pronton motive force (PMF). ATP is a universal energy currency in all forms of life. PMF is the combination of electrical and chemical potential difference across bacterial cell membrane. In this thesis, we use the fluorescent protein (QUEEN-QUantitative Evaluator of cellular ENergy)[1] and the bacterial flagellar motor (BFM) to probe the ATP and PMF respectively. QUEEN sensor is a circularly-permuted ATP sensitive green fluorescent protein. BFM is the molecular motor driven by ion flux and PMF. By these two cutting-edge biophysical probes, we could measure the dynamics of these two main energy source in a bacteria cell.
We aim to measure the ‘free energy’ of a bacterial cell could utilize under extreme starvation. Besides, we also want to know if we could starve a bacterial cell to death. After Escherichia coli cells being transferred from rich medium to zero nutrient medium, the BFM rotation speed drops exponentially with a load-dependent decay rate. The total energy consumed through BFM while starvation is roughly on the scale of mM ATP, equivalent to 10-13 Joule per E. coli and is load–independent. After BFM speed and PMF drop to zero, the cells are retreated with rich medium. Surprisingly, most bacterial cells are viable and can recover from the starvation. The single cell measurement of intracellular ATP by QUEEN fluorescent protein shows the same order of energy before starvation. The measurements shed a new way to probe bacterial energetics and dynamical response under starvation.

關鍵字(中)

★ 細菌鞭毛馬達
★ 三磷酸腺?
★ 大腸桿菌

關鍵字(英)

★ Bacterial Flagellar Motor
★ Adenosine Triphosphate
★ Escherichia coli

論文目次

1 INTRODUCTION 1
1.1 MOTIVATION 1
1.2 ENERGETIC OF E. COLI 2
1.3 MAINTENANCE ENERGY 3
1.4 STARVATION WITHOUT NUTRIENT 4
1.5 PROTON MOTIVE FORCE (PMF) 5
1.5.1 Bacteria Flagellar Motor (BFM) 6
1.5.2 Relation between PMF & BFM 7
1.6 ADENOSINE TRIPHOSPHATE (ATP) 8
1.6.1 QUEEN Fluorescent Protein 8
1.6.2 In vivo ATP measurement 9
2 MATERIAL AND METHODS 10
2.1 BACTERIAL STRAINS 10
2.2 MEDIUM 11
2.3 OPTICAL DENSITY SPECTROPHOTOMETRY 12
2.4 FLUORESCENCE MICROSCOPY 13
2.5 TECHNIQUE 14
2.5.1 Tethered Cell Assay 14
2.5.2 Bead Assay 15
2.5.3 Spread Plate 16
2.6 PMF MEASUREMENTS 17
2.6.1 Bead Assay with Continuous Flow 17
2.6.2 Starvation & Recovery 18
2.6.3 Instruments and Analysis 19
2.6.3.1 Microscope 19
2.6.3.2 CCD-GE680 20
2.6.3.3 Speed Analysis 20
2.7 ATP MEASUREMENTS 22
2.7.1 QUEEN Fluorescent Experiment 22
2.7.2 In vitro Calibration 22
2.7.3 In vivo ATP Measurement 22
3 RESULTS 23
3.1 PMF MEASUREMENTS 24
3.2 PMF MEASUREMENT - STARVATION 25
3.2.1 PMF Decaying with Different Load 26
3.2.2 Energy Consumed through BFM 27
3.3 PMF MEASUREMENT - RECOVERY 28
3.3.1 Survival Probability w/ | w/o PMF 28
3.3.2 Death Rate by Spread Plate 28
3.4 ATP MEASUREMENTS 31
3.4.1 In vitro Calibration 32
3.4.2 In vivo ATP Measurement 33
4 DISCUSSION & OUTLOOK 34
4.1 ENERGY REQUIREMENT OF A BACTERIA CELL 34
4.2 STARVATION WITH/WITHOUT NUTRIENT 35
4.3 OUTLOOK 36
5 BIBLIOGRAPHIES 37
6 APPENDICES 39
6.1 ABBREVIATIONS 39
6.2 APPENDIX A (PMF MEASUREMENTS PROTOCOL) 40
6.3 APPENDIX B (ATP MEASUREMENTS PROTOCOL) 41
6.4 APPENDIX C – FLUORESCENCE IMAGING FILTERS 43

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指導教授

羅健榮(Chien-Jung Lo)

審核日期

2018-7-24

推文