Quantitative Study Of The Relationship Between Bacterial Cell Cycle And Its Key Proteins Via Flow Cytometry

The bacterial cell cycle and its key proteins regulate the growth and division of bacteria.Clarifying the relationship between the two is of great significance for promoting basic microbial research and the development of new antibacterial drugs to resist the increasing threat of bacterial resistance.The key proteins of the bacterial cell cycle are highly individual and diverse in time and space.Their expressions sometimes are extremely low and closely related to other biomolecules.Therefore,how to reveal the relationship between cell cycle and key proteins at the singlebacteria level,and achieve simultaneous analysis with other biological indicators is the problematic point at this stage.In order to solve the research difficulties at this stage,this paper used the advantages of flow cytometry which is single-bacteria level,high-throughput and multi-parameter detection,combined with nucleic acid labeling technology and biarsenical-tetracysteine labeling technology,developed an approach for quantitative multi-parameter analysis of bacterial cell cycle-related parameters(nucleic acid,protein expression,and cell length)at the single-bacteria level,and the correlation between the various parameters was obtained.This dissertation consists of the following sections:The first chapter is a literature review.We made an overview of the bacterial cell cycle and the main functions of key proteins,as well as the recent advanced characterization technology.The motivation and research content was also included in this part.The second chapter introduces the establishment of a flow cytometric method for the detection of bacterial cell cycle parameters.Combined with nucleic acid dye labeling and software fitting,the cell cycle parameters such as the start time and duration of B,C,and D phases of E.coli MG 1655 under different nutrient conditions were achieved,and the regularity of the C phase duration of fast-growing bacteria was found.The third chapter is the establishment of an in situ flow cytometric analysis of the FtsZ protein.As a key protein in the bacterial division,FtsZ protein has great application potential in the development of new antibacterial drugs.Using the biarsenical-tetracysteine(FlAsH-TC)system for the in-situ labeling of FtsZ,we detected the expression of FtsZ protein in E.coli MG1655 under various nutritional conditions and the expression of FtsZ protein in a cell cycle.Moreover,we found that the expression of FtsZ protein was positively correlated with the degree of nutrition,and the expression of FtsZ protein was up-regulated and then down-regulated in a cell cycle under slow-growth conditions.Chapter four is the construction of a multi-parameter detection method for key players in the bacterial cell cycle.Based on the performance of simultaneous detection of scattered light and multi-fluorescence by flow cytometer,we achieved simultaneous detection of the DNA content,FtsZ protein expression and cell length at the single-bacteria level.The positive correlation between DNA content,FtsZ protein expression and cell length at the single-bacteria level was found for the first time.The developed method will provide advanced technology for the basic research of the bacterial cell cycle.In the last chapter,the research progress was summarized and the prospects of bacterial cell cycle were discussed.