| The organism is an important part of nature.Although different creatures have different structural traits and functions,the basic unit of their life is the same.Therefore,in a sense,they may follow similar or even the same basic laws.Following the basic development model of scientific research,we selected Escherichia coli,a simple single-cell prokaryotic bacteria,as the research object.Researchers like to use them as research model for their simple structure,easy access,easy cultivation,rapid breeding and other characteristics.Unlike the behavior of the object at the macroscopic scale,the micron sized E.coli is like a fine small machine that performs some basic functions of the creature,such as chemotaxis,at the micron scale.In this paper,the adaptive signals of E.coli flagellar motor and internal chemotactic signal protein output were observed and recorded by means of latex beads labeling and fluorescence resonance energy transfer.Using the basic method of signal processing,a simple mathematical physics model is used to explain the differences in adaptive signals caused by various conditions.The effects of signal differences on the chemotaxis of E.coli were described by experiments and simulations.In the following,we will briefly studied the main contents of each chapter.The introduction of this paper is mainly about the basic facts of E.coli and its related research progress.We began the introduction of E.coli with its shape and structure,followed by genes,proliferation and pathogenicity.The focus is on motility and chemotaxis which are the two major research areas of E.coli.For the description of motility,we introduced the structure and composition of the various moving parts at first,including the flagellar motor which was embedded in three-layers wall of cells,the hook working as the transmission part,and the terminal output called bacterial flagella.The most important part of them is flagellar motor which is source of movement.Secondly,we briefly described the swimming in a liquid environment,swarming on the gel surface,and the results of swarming-biofilm' formation.For the introduction of chemotaxis,We have described the receptors and chemotaxis signal transduction pathway in detail.Then we summarized the two mainstream models,MWC model and Ising model,for the chemotactic network and flagellar motor of E.coli,At the end of the introduction,we summarized the significance and main contents of this paper.The second chapter of this paper is a brief introduction of the experimental methods.At the beginning of this chapter,we briefly described the methods and applications of various reagents.Followed by a brief explanation of the principles and procedures for the DNA recombination technology we have used,including transformation,electroporation,polymerase chain reaction and ? Red deletion.The third chapter is about exploring the effect of growth conditions on the chemotaxis of E.coli.We have measured the growth curve under poor and rich nutrient cultures(HI and TB)respectively,and selected the appropriate growth stage for observation.The CW bias distribution,the motor correlation time in the absence of stimulation,the recovery time under a step stimulus of 50 mM MeAsp,and the aggregation in the capillary experiments of the two kinds of cells have been compared.It was found that the CW bias distribution of them were roughly the same,and both the correlation time and recovery time showed a great difference.The cells grown in the HI solution had a shorter correlation time and recovery time,which were 1.65 and 1.36 times of TB cells,respectively.Finally,we demonstrated that the E.coli grown in H1 perform stronger localization for stimulus by capillary assays and simulations,and they were able to respond more quickly to changes in the environment to complete the chemotaxis.The fourth chapter is the work about the effect of receptors interaction on bacterial chemotaxis.We used the beads assays and FRET technique to measure the step response signal and the dose response curve of the mutant strain with single type of receptor and the wild type strain.We also changed the ratio of Tar and Tsr receptors to observe their difference in step response.Finally,a simple swim plate experiment was used to compare the chemotactic ability.The results showed that the mutant strain performed an obviously partial-adaptation behavior when the concentration of MeAsp was in the range of 5?M to 250 ?M,while the wild type strain could adapt almost perfectly.We found that the adapted activity and adaptation time of the mutant strain change significantly with the stimulus concentration,while the changes of the wild type strain are much smaller.After comparing their dose-response curves,we obtained the total number of receptor dimers in a ?MWC cluster in the mutant strain was about 11.7,while it was about 19.5 in the wild type.By introducing a function related to the methylation level,we have got the methylation rate of the mutant cells that is about 0.56 times the wild type by simulation,which should be related to the difference in the receptor cluster.In addition,we also studied the effect of receptor ratio on the overshoot.Finally,swim plate experiments confirmed that the chemotaxis of the mutant bacteria was worse.The fifth chapter is the work about building an analytical model of motor adaptive remodeling.Bacterial chemotaxis is a canonical system for the study of signal transduction.One of the hallmarks of this system is its robust adaptive behavior.However,how fast the system adapts remains a controversy.The adaptation time measured at the level of the kinase activity was tens of seconds,while that measured at the level of flagellar motor was less than ten seconds.The flagellar motor was shown recently to exhibit adaptive remodelling,its main physiological function being to provide a robust match between the receptor output and motor input,and its adaptation timescale thought to be too slow to contribute much to the overall adaptation timescale of the chemotaxis system.Here,through theoretical modeling of the motor adaptive remodelling and experimental tests,we showed that this partial adaptation contributes significantly to speed up the adaptation,thereby resolving the previous controversy.The sixth chapter is the work about summaries and outlook of the three core work.The first work which was mainly based on beads assays and computer simulations showed that E.coli growing in H1 adapted faster.The second work was to study the effect of receptor synergy on the chemotaxis of E.coli with beads assays and FRET experiments.We found that the mutant strain with single type of receptor showed worse accuracy,but could show overshoot behavior which was similar to the wild type.The third work was to build an analytical model of motor adaptive remodeling.The experimental model successfully explained the previous controversies about the adaptive time measurement results.And the outlook was mainly about the problems that were still unresolved in the article.For example,the interpretation of partial adaptation of the single type receptor strain and the further improvement of motor adaptive remodeling model,and so on. |
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