Gene Regulation in Phage Lambda: A Real-Time Study with Single-Event Resolution

噬菌体 Lambda 中的基因调控：单事件分辨率的实时研究

基本信息

批准号：
9113607
负责人：
Ido Golding
金额：
$ 29.74万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-04 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9113607
关键词：
Address Algorithms Bacteria Bacteriophage lambda Bacteriophages Beds Binding Biochemical Cell Death Cells Cereals Cytolysis Cytoplasm DNA Data Analyses Dependence Diffusion Disease Dosage Compensation (Genetics)Escherichia coli Event Fluorescence Microscopy Funding Gene Dosage Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Genomics Goals Health Heterogeneity Human Image Analysis In Vitro Individual Infection Kinetics Knowledge Life Life Cycle Stages Lysogeny Maps Measures Memory Messenger RNA Methods Microscopic Modeling Mono-S Noise Organism Outcome Phenotype Play Production Property Proteins RNA Regulation Resolution Role Series Shapes System Testing Theoretical model Time Time Study Transcriptional Regulation Viral Genome Virus Work base biochemical tools biophysical tools cell behavior chemical reaction discrete time dynamic system improved mathematical model particle promoter research study single molecule spatiotemporal tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): The system comprising the bacterium Escherichia coli and its virus, bacteriophage lambda, has long served as a simple paradigm for the way gene regulation drives the choice between alternative cellular states, the inheritable memory of cell state, and the switching from one state to another. The lambda system has been extensively characterized using genetic and biochemical approaches. More recently, it has served as one of the first test beds for the attempt to form a quantitative narrative for a living system, in the shape of mathematical models connecting the microscopic physical-chemical reactions in the cell to the system-level properties. However, these models still have limited predictive power, due to the absence of an experimentally- based description of gene regulation at the required spatiotemporal resolution. Our goal in this competitive renewal is to continue closing this knowledge gap by quantifying gene regulation in the lambda system at the resolution of individual phages and cells, individual gene copies in the cell, individual molecules and discrete events in space and time. To achieve this goal, we will use single-cell and single-molecule fluorescence microscopy, which, combined with advanced image and data analysis algorithms, allow us to detect individual phage particles and individual molecules of DNA and RNA, count absolute protein numbers in individual cells and measure the discrete time-series of transcription. By using simple, coarse-grained theoretical models we are able to distill our experimental findings into general principles, which provide an improved system-level understanding of lambda, and can be directly applied to findings in higher systems. The outcome of the proposed work will be a quantitative description of gene regulation at the cellular, "mesoscopic" scale, providing a bridge between the two currently-existing levels of description: the microscopic details of molecular interactions governing gene regulation, obtained using traditional biochemical and biophysical tools in vitro, and large scale ("macroscopic") topologies of gene networks, mapped using genetic and genomic methods. Specifically, the work will allow us to address the following questions: (1) To what degree is the observed heterogeneity ("noise") in gene regulation a manifestation of actual biochemical stochasticity, or instead represents our inability to measure cellular "hidden variables", which have a deterministic effect on cell behavior? (2) What role do spatial effects, beyond simple diffusion in a homogenous cytoplasm, play in gene regulation? Ultimately, the conceptual and experimental tools developed in this work will further our understanding of how gene regulation drives cell-fate choices in higher, multicellular systems, and in the context of human health and disease

描述（由申请人提供）：包含大肠菌及其病毒及其病毒的系统，长期以来一直是基因调节的方式的简单范式，以驱动替代性细胞状态，可遗传的细胞状态以及从一个状态转换为另一种状态之间的选择。使用遗传和生化方法对Lambda系统进行了广泛的表征。最近，它是试图形成生命系统定量叙事的第一批测试床之一，它以数学模型的形式连接了细胞中的微观物理化学反应与系统级属性。但是，由于缺乏基于实验的基因调节描述，这些模型仍然具有有限的预测能力。我们在这种竞争更新中的目标是通过量化单个噬菌体和细胞的分辨率，通过量化lambda系统中的基因调节来继续缩小该知识差距和时空中的离散事件。为了实现这一目标，我们将使用单细胞和单分子荧光显微镜，该显微镜结合了先进的图像和数据分析算法，使我们能够检测到单个噬菌体颗粒和DNA和RNA的单个分子，计数单个细胞中的绝对蛋白质数，并测量转录的离散时间序列。通过使用简单，粗粒的理论模型，我们能够将我们的实验发现提炼为一般原则，从而提供了对Lambda的系统级别的理解，并且可以直接应用于高级系统中的发现。拟议工作的结果将是对细胞“介镜”量表基因调控的定量描述，在两个当前存在的描述级别之间提供了一个桥梁：使用传统的生物学和生物物理学工具在VITRO和GLAND SCON DOBLODISS和GLAND SCOLSIDICE中获得的分子相互作用的显微镜细节，该基因调节的微观细节（”基因组方法。具体而言，这项工作将使我们能够解决以下问题：（1）基因调节中观察到的异质性（“噪声”）的程度在多大程度上是实际生物化学随机性的表现，或者代表我们无法测量细胞“隐藏变量”，哪些对细胞行为具有确定性影响？（2）在基因调节中，空间效应超出了均质细胞质中的简单扩散何种作用？最终，这项工作中开发的概念和实验工具将进一步了解基因调节如何推动更高，多细胞系统的细胞污染选择，以及在人类健康和疾病的背景下