Probing bacteria single-cell membrane potential by a genetically encoded voltage indicator

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莊蕎蓁(Chiao-Chen Chuang) 查詢紙本館藏

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物理學系

論文名稱

(Probing bacteria single-cell membrane potential by a genetically encoded voltage indicator)

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至系統瀏覽論文 (2026-8-1以後開放)

摘要(中)

一般人認知細菌的膜電位是很穩定的。近幾年科學界發現細菌細胞的膜電位是動態的，並且具有多種功能。這引發了對細菌膜電位更深入洞察的興趣。然而，由於細菌體積小且具有細胞壁，測量其膜電位具有挑戰性。此外，使用分子染料可能導致訊號變得更加複雜。因此，開發了一種名為ViBac的新型基因編碼電壓指示方法，將螢光蛋白與電壓敏感的感測器結合，作為膜電位的指示劑。

本研究探索了細菌在表面感知和適應反應過程中單個細胞和群體水平的膜電位變化。利用ViBac感測器，在單個細胞中觀察到了超極化尖峰事件，持續了1分鐘的時間序列。研究分析了這些膜電位變化，這些變化歸因於電荷分離的差異，在不同的環境條件下觀察到了尖峰行為。同時，對這些尖峰現象進行了詳細的特性化研究，以增進對細菌膜動態和適應反應的理解。

膜電位在細胞分裂等基本細胞過程中起著至關重要的作用。因此，我們還研究了細胞分裂期間膜電位的變化。通過使用創新的螢光指示技術和不同的實驗條件，我們觀察到了細菌在不同狀態下的膜電位動態。

摘要(英)

It is a long belief that the membrane potential of bacterial cells is static. Recently, it has been discovered that the bacterial membrane potential is dynamic and multifunctional. This has sparked interest in gaining deeper insights into bacterial membrane voltage. However, measuring membrane voltage in bacteria is challenging due to their small size and the presence of a rigid cell wall. Additionally, the use of molecular dyes can result in more complex signals. Thus, a new type of Genetically Encoded Voltage Indicator (GEVI) method called ViBac has been developed, which combines fluorescent proteins with voltage-sensitive sensors to serve as indicators for membrane potential.
This study explores changes in membrane potential at both the single-cell and population levels in bacteria during surface sensing and adaptation responses. Utilizing the ViBac sensor, hyperpolarization spiking events were observed over a 1-minute time series in individual cells. The variations in membrane voltage which are attributed to differences in charge separation were examined under different environmental conditions to observe spiking behaviors. Detailed characterization of these spiking phenomena was also performed to enhance the understanding of bacterial membrane dynamics and adaptive responses.
Membrane voltage plays a crucial role in fundamental cellular processes such as cell division. Therefore, we also investigated changes in membrane voltage during the cell division period. By employing an innovative fluorescent indicator technique and varying experimental conditions, we show the membrane voltage dynamics of bacteria across different states.

關鍵字(中)

★ 細菌膜電位
★ 基因編碼電壓指示
★ 細胞分裂
★ 細胞表面感應

關鍵字(英)

★ Bacterial membrane potential
★ Generically encoded voltage indicator
★ cell division
★ surface sensing

論文目次

摘要.................................. i

Abstract............................ii

Acknowledgement ...........iii

Table of Contents ............iv

List of Figures ..................vi

List of Tables....................ix

1 Introduction....................................... 1

1.1 Cell Membrane .................................. 1

1.2 Membrane Potential................................ 2

1.3 Membrane Transportation............................. 3

1.4 Action Potential.................................. 3

1.5 Measurement of Membrane Potential....................... 7

1.5.1 Patch clamps ............................... 7

1.5.2 Nernstian dyes .............................. 8

1.6 Genetically Encoded Fluorescent Proteins .................... 9

1.7 Bacterial Surface Sensing and Response..................... 10

2 Materials and Methods................................. 12

2.1 Voltage Indicator for Bacteria(ViBac) ...................... 12

2.1.1 Function of Arclight and mCherry.................... 12

2.1.2 Type and Structure of ViBac....................... 13

2.2 Bacterial strain .................................. 14

2.2.1 Arabinose................................. 15

2.2.2 Chloramphenicol............................. 15

2.3 Growth Condition................................. 16

2.3.1 Medium and Buffer............................ 16

2.3.2 Growth Environment........................... 16

2.4 Experiment Condition............................... 17

2.4.1 Gene Frame & Gel Pad.......................... 17

2.5 Apparatus..................................... 18

2.5.1 Microscope................................ 18

2.5.2 Phase Contrast .............................. 20

2.5.3 Perfect Focus System........................... 21

2.6 Image analysis .................................. 22

3 Results.......................................... 24

3.1 Cell Spiking.................................... 24

3.1.1 Hyperpolarization ............................ 25

3.1.2 Ion Channel................................ 26

3.1.3 Spiking Events . . . . . . . . . . . . . . . . . 27

3.1.4 Spiking Characteristics .......................... 33

3.2 ViBac Membrane Voltage Calibration ...................... 36

3.3 Change in Vm during Cell Division ........................... 40

4 Discussion........................................ 48

References ......................................... 50

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指導教授

羅健榮(Chien-Jung Lo)

審核日期

2024-7-29

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