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Undergraduate Summer Research Experience: Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

本科暑期研究经历：原核生物模型中昼夜节律的分子和细胞机制

基本信息

批准号：
10810593
负责人：
SUSAN S GOLDEN
金额：
$ 1.3万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-04 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10810593
关键词：
Address Anacystisnidulans Animals Bar Codes Binding Biochemical Biological Biological Assay Biological Clocks Biological Models Biological Rhythm Biotechnology Cardiovascular Diseases Cell physiology Cells Circadian Dysregulation Circadian Rhythms Clock protein Complex Cryo-electron tomography Cues Cyanobacterium Cytology Disease Environment Escherichia coli Eukaryota Event Functional disorder Genes Genetic Genomics Growth Health Human In Vitro Ions Libraries Malignant Neoplasms Mammals Mediating Mental disorders Metabolic Metabolic syndrome Metabolism Modeling Molecular Nucleotides Organism Personal Satisfaction Phase Phenotype Phosphotransferases Phylogenetic Analysis Physiological Physiology Preparation Production Prokaryotic Cells Property Protein Biochemistry Proteins Regulation Resolution Sasa Signal Transduction Site Sleep Disorders System Time Trees Visualization Work circadian circadian pacemaker experience experimental study fitness model organism pathogen promoter reconstitution summer research transcription factor undergraduate student

项目摘要

This project leverages a cyanobacterial model system to answer the following questions: what are the molecular interactions that mark the passage of time in a cell, where do they occur in the cell, how do they mediate temporal regulation of events, and why does biological timing matter for fitness? The circadian biological clock is an oscillatory timer that drives 24-h rhythms of biological activities. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. However, the circadian clock is pervasive well beyond mammals, promoting fitness in diverse organisms throughout the phylogenetic tree. The circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. A new in vitro preparation comprising the oscillator proteins KaiA, KaiB, and KaiC, along with the kinases CikA and SasA and the transcription factor RpaA, reconstitutes the circadian rhythm of binding of RpaA to its target promoter with a real-time readout. This project will apply the in vitro clock and other technical and conceptual advances towards biochemical, cytological, genomic, and physiological objectives that will answer the target questions. The in vitro clock will reveal the molecular events that occur when the clock resets to an environmental timing cue, identify the sites of action of nucleotides that modulate the timing circuit, and determine how RpaA and a second transcription factor that is regulated by environmental signals, RpaB, work together to influence circadian phasing. The discovery that the kinases SasA and CikA impart tolerance to fluctuating oscillator component concentrations will overcome past hurdles for establishing a circadian circuit in Escherichia coli as a naïve model system for exploring clock connections to cellular physiology and for biotechnology applications. High-resolution cryo-electron tomography and focused ion-beam milling will be used to visualize clock-controlled daily changes in intracellular organization and the clock complex itself. The molecular basis and fitness advantage of circadian control of natural transformation will be determined. A bar-coded transposon library first used to identify all genes required for photoautotrophic growth will be used to identify new loci that contribute to fitness in a day-night cycle. Paired with physiological and metabolic assays, these experiments will answer the question: why does the timing of molecular events matter? Together, these approaches will elucidate clock mechanisms and the value of the clock to diurnal physiology, and will advance biotechnological opportunities for controlling metabolism in both photosynthetic and traditional bacterial production systems.

这个项目利用蓝藻模型系统来回答以下问题：什么是分子