【作者】 王彬；

【导师】 袁军华；

【作者基本信息】 中国科学技术大学 ， 凝聚态物理， 2018， 博士

【摘要】 虽然细菌是最古老的生命体之一,但是它与我们的生活是息息相关的,从现代医学观点来看细菌的致病性仍是我们日常生活中产生疾病的重要来源之一。随着现代医学的发展,抗生素的问世解决了大部分由于细菌引起的疾病,但是仍发现有些超级细菌顽固地存在于含有抗生素的环境中,这又给我们带来了新的难题。虽然现在对于细菌致病的过程有了一定程度上的研究,但是关于细菌具体的致病机制仍然是亟需解决的问题。如果能够阐述清楚细菌的致病机制,我们可以从本源上理解细菌的致病过程,这样就可以开发新的药物或者新的治疗过程。由于细菌的运动行为与致病性是紧密联系在一起的,这又让我们重新回到了最原始的问题一细菌运动行为的机制究竟是如何?上个世纪越来越多的生物学家、物理学家尝试通过实验设计和理论建模来研究这个问题,取得一系列重要的进展。本文研究也隶属研究细菌运动行为的行列之中,将从四个部分进行介绍。第一章绪论部分将对近几十年关于大肠杆菌的系列研究进行介绍。具体介绍了大肠杆菌的细胞结构,个体的游动,趋化网络的实验研究和模型研究,鞭毛勾的柔韧性,鞭毛马达运动的动力来源、马达的步进行为、马达部件MotB的动态交换方式、马达的力矩-速度曲线,马达上定子的力学敏感性及其可能来源,本文的研究内容等等。第二章我们研究了马达占空比的问题。近二十年来,关于马达占空比的问题一直在实验上和理论上争论不断,一直没有直接的实验证据证明这个问题。我们发展光镊、激光暗场与明场成像结合技术,基于“bead addition”观念,从实验上验证在零负载下鞭毛马达转动速度与其上绑定的定子个数无关,进而证明马达的占空比为1。对于马达占空比的测量,可以排除现在理论上关于马达的一些假定,让马达模型的物理图像越来越明晰。而且我们提供了一种精确测量在零负载下鞭毛马达上绑定定子个数的方法,这种方法解决了一直无法精确测定当较少数目的定子绑定在马达上时的定子数目问题。在光学技术发展方面,我们实现了高时间分辨率下的纳米金球的可视化并且同时可以运用光镊技术,为以后研究光与纳米金球相互作用提供了一种实验上的可能性。第三章我们主要研究“马达的质子输运与马达的旋转是紧耦合还是松耦合”的问题。大肠杆菌鞭毛马达在高负载的情况下被认为力矩输出的效率达到最大,但是关于马达在高负载下转动一周需要消耗的质子数仍然没有很准确的数值,因为目前不能精确测量出马达在高负载下的力矩。但是根据力矩-速度曲线的走势,可以近似认为马达在高负载下转动时的输出力矩与马达在被拉停时的输出力矩(也就是静止力矩)相等,所以第三章主要的目的是讲述如何精确测定鞭毛马达的静止力矩。其中前一部分主要讲述几种鞭毛马达静止力矩的测定方法、单一用磁镊来测定鞭毛马达的静止力矩以及关于这种方法测定过程中可能引入的不准确性,后一部分我们主要讲述如何消除这种不准确性的方案,比如在磁镊测量的基础上引入光镊或其它技术等等。尝试用磁镊去测量鞭毛马达的静止力矩,从实验上去证明马达的质子输运与马达的旋转究竟是是紧耦合还是松耦合。第四章我们主要对以上的工作进行小结和结合这几年自己的工作与前人的工作对接下来的工作进行展望。第一项工作主要测定了大肠杆菌马达的占空比为1,解决了先前的争端,明晰了马达运动的物理图像。第二项工作主要介绍了一种实验上可能精确地测定大肠杆菌静止力矩的方法,以及现在遇见的一些问题,和针对解决这些问题未来实验上的改进。最后结合以上的工作和先前的工作,可以用我们新发展光镊、激光暗场与明场成像结合技术对之前大肠杆菌运动行为并不太精确的测量进行重新测量,比如说在不同定子数目的力矩-速度曲线等等;同时如果我们可以把磁镊用于大肠杆菌的测量中,这样就可以运用大肠杆菌力矩输出的性质来反向研究磁镊的一些性质,丰富关于磁镊力矩产生的信息,推动磁镊在其他领域的应用。更多还原

【Abstract】 Although bacteria is one of the oldest living organisms,the pathogenicity of bacteria is still one primary source of disease in our daily life from the view of modern medicine.The advent of antibiotics has solved most of the diseases caused by bacteria as modern medicine advanced.However,some superbacteria still exist the environment with antibiotics,leading to new problems about bacterial pathogenesis.Having been studied to some extent in past several decades,the specific pathogenic mechanism of bacteria,which is closely related to the motile behavior of bacteria,is still in urgent need to be solved.If these mechanisms could be well understood,new drugs or new treatment processes would be developed.Therefore,the most primitive question of how the mechanism of the movement of bacteria is operating was brought back to our view.In the last century,a series of important progresses on experimental design and theoretical modeling for studying this problem have been made by more and more biologists and physicists,and this study also belongs to the study of the behavior of bacteria,which will be divided into four chapters.Chapter one will introduce a series of studies on Escherichia coli in the last few decades,referring to the cell structure of Escherichia coli,individual swimming behavior,experimental and modeling studies of chemotaxis network,the flexibility of flagellar hook,the source of motor motion,flagellar motor stepping motion,MotB(motor component)exchange,the torque-speed curve of the motor,the dynamic nature of mechanical sensitivity,the research content of this paper and so on.In the second chapter,we mainly study the duty ratio of the motor,which has been debated both experimentally and theoretically in the past twenty years,and there has been no directly experimental evidence to solve this argument.We developed a technique combining optical tweezer,laser dark field and bright field imaging.We first used bead addition technique to measure the number of stators binding to motor at zero load,proving that the zero-load speed is independent of the stator number,thus determining that the duty ratio is close to 1.Therefore,some theoretical assumptions on the flagellar motor would be eliminated,the physical image of the motor becoming clearer and clearer.In summary,we developed a method to precisely measure the stator number for flagellar motor at zero load.This method could also solve the problem that the number of stators binding to motor is so few that this number would be unable to be determined previously.In terms of technological development,we realized the visualization of nano gold spheres at high temporal resolution and applied optical tweezers technology simultaneously,which provided an experimental possibility for future research on the interaction between optics and gold sphere.In the third chapter,we study the problem that the proton transport of the motor and its rotation is tightly or loosely coupled.Genally speaking,the efficiency of the flagellar motor of Escherichia coli at high load would reach the maximum,but number of protons consumed when the motor steps one revolution,is still not very precisely determined because the motor torque at high load is not measured accurately.But according to the trend of torque-speed curve,it could be assumed that the output torque of the motor is approximately equivalent to the torque when the motor is pulled and halted externally(stall torque).Therefore,the first part of this chapter is to on how to accurately measure the stall torque of the flagellar motor,including several early works on measuring the stall torque.Magnetic tweezer would be a suitable technique to measure the stall torque of flagellar motor,but the process of calibrating the torsional stiffness of magnetic tweezers is still unaccurate.In second part,we mainly talk about how to eliminate the uncertainties,such as by combining with optical tweezers or other techniques.In a word,we try to use magnetic tweezers to prove whether the motor’s proton transport is tightly or loosely coupled with the rotation of motor experimentally.In the fourth chapter,based on our own work and the previous work in the past few years,we mainly summarize the above work and look forward to the future work.The first work was determining that the duty ratio of the Escherichia coli motor is close to 1,resolving the previous dispute and clarifying the physical images of the motor movement.The second work mainly introduced a possible way to accurately measure the stall torque of Escherichia coli,some of the problems we met now,and future improvements to solve these problems.Finally,considering the above work and previous works,we could use our newly developed technology combining optical tweezer,laser dark-field and bright-field imaging to more accurately study the properties of the flagellar motor,such as the torque-speed curve for motors with a specific number of stators and so on;in addition,we introduced magnetic tweezer for measurement of stall torque of Escherichia coli,and if succeeded,conversely,certain properties of magnetic tweezer could be investigated with the output torque of the flagellar motor,thus promoting the application of magnetic tweezer to other areas.更多还原