Dynamical interrogation of the Bacillus subtilis sporulation network using an engineered light-switchable promoter system

使用工程光开关启动子系统动态询问枯草芽孢杆菌孢子形成网络

• 批准号: 9059017
• 负责人: Jeffrey Jay Tabor
• 金额: $ 18.66万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9059017
• 关键词: Acute; Animal Model; Anti-Bacterial Agents; Asthma; Autoimmune Diseases; Bacillus anthracis; Bacillus cereus; Bacillus subtilis; Bacteria; Biochemical; Biological; Biological Process; Calcium; Cell Cycle; Cells; Chemicals; Chromosomes; Clostridium difficile; Controlled Study; DNA; DNA biosynthesis; Decision Making; Defect; Development; Distant; Engineering; Ensure; Escherichia coli; Event; Feedback; Frequencies; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Growth; Health; Human; Hypersensitivity; Individual; Infection; Inherited; Life; Light; MAP Kinase Gene; Malignant Neoplasms; Methods; Microfluidics; Modeling; Optical Methods; Outcome; Pathway interactions; Phase; Phenotype; Phosphotransferases; Physiologic pulse; Physiological; Play; Population; Process; Property; Proteins; Ramp; Regulator Genes; Reporting; Repression; Reproduction spores; Resistance; Role; Saccharomyces cerevisiae; Series; Sigma Factor; Signal Transduction; Signaling Protein; Starvation; Stress; Synthetic Genes; System; Technology; Time; Work; base; biological adaptation to stress; design; extracellular; gene product; genetic regulatory protein; improved; information processing; innovation; insight; mathematical model; microbial; new technology; novel; novel therapeutics; optogenetics; pathogen; premature; programs; promoter; protein-histidine kinase; response; segregation; sensor; success; time use; tool; transcription factor

 DESCRIPTION (provided by applicant): The ability to understand and manipulate the biochemical events controlling how cells make decisions is central to the development of novel treatments for microbial infections, autoimmune diseases, cancer, and developmental defects. The bacterium Bacillus subtilis differentiates into stress-resistant, metabolically inert spores upon starvation, and activates a separate gene general stress response pathway when challenged with various stresses. B. subtilis sporulation and stress response are ideal model pathways for which to develop innovative new technologies to study and control differentiation. Though the basic regulatory interactions in the core gene circuits are known, we lack a systems-level understanding of how dynamical changes in the major regulatory proteins result in cellular information processing and the ultimate guide these large-scale cellular decisions. Our central hypothesis is that the ability to dynamically perturb and observe protein activities in gene circuis in real time will yield crucial insights about cellular decision-making and differentiation. To thi end, we propose to develop a technology for interrogating the signaling properties of the B. subtilis sporulation and stress response gene circuits that uses time-varying light signals to program exceptionally well-defined gene expression dynamics in live cells. As a first aim, we will re-engineer a green/red light-switchable two-component system we previously built in E. coli to control transcription and generate dynamical gene expression functions in B. subtilis. By moving this system from E. coli to the evolutionary distant B. subtilis, we will also gain insights on the considerations needed to move these powerful optogenetic tools to other model organisms and clinically important species. As a second aim, we will use our optical method to analyze how different rates of activation of the major sporulation regulators impact the circuit functions and resulting phenotype. We expect to unequivocally demonstrate that i) successful execution of the sporulation program requires not only proper steady-state levels, but also proper dynamics of key regulators and that ii) recently observed pulsing in the major stress response regulator are used to elicit proportional activation levels of many target genes. In the third aim, we will use or optical method to investigate the biological significance of the recently described pulsatile dynamics of the master sporulation regulator at the population and single cell levels. In particular, we will evaluate our recent hypothesis that the sporulation pulses must occur after DNA replication to ensure that spores inherit chromosomes and a prevailing hypothesis that a supra-threshold concentration of the master sporulation regulator must be reached for cells to commit to sporulation. Since the B. subtilis sporulation and stress response circuits are widely conserved among medically important spore-forming bacteria including B. cereus, B. anthracis, and C. difficile, our result will not only enable breakthroughs in the understanding of cellular decision-making, but also provide a basis for design of new antibacterial agents.

 描述（由申请人提供）：理解和操纵控制细胞如何做出决定的生化事件的能力对于开发微生物感染、自身免疫性疾病、癌症和发育缺陷的新型治疗方法至关重要。枯草芽孢杆菌在饥饿时分化成抗应激的代谢惰性孢子，并且在受到各种应激的挑战时激活单独的基因一般应激反应途径。B。枯草芽孢杆菌孢子形成和应激反应是理想的模型途径，用于开发创新的新技术来研究和控制分化。虽然核心基因回路中的基本调控相互作用是已知的，但我们缺乏对主要调控蛋白的动态变化如何导致细胞信息处理以及最终指导这些大规模细胞决策的系统水平的理解。我们的中心假设是，动态扰动和观察蛋白质活动的能力，在真实的时间基因circuis将产生至关重要的见解细胞决策和分化。为此，我们建议开发一种用于询问B的信令特性的技术。枯草芽孢杆菌孢子形成和应激反应基因电路，其使用时变光信号来编程活细胞中异常明确的基因表达动力学。作为第一个目标，我们将重新设计一个绿色/红色光可切换的双组分系统，我们以前建立在E。在B. coli中控制转录并产生动态基因表达功能。枯草杆菌。通过将该系统从E.大肠杆菌与进化远缘的B。枯草杆菌，我们还将深入了解 需要考虑将这些强大的光遗传学工具转移到其他模式生物和临床重要物种。作为第二个目标，我们将使用我们的光学方法来分析主要孢子形成调节剂的不同激活速率如何影响电路功能和由此产生的表型。我们期望明确地证明i）孢子形成程序的成功执行不仅需要适当的稳态水平，而且需要关键调节剂的适当动力学，以及ii）最近观察到的主要应激反应调节剂中的脉冲用于引发许多靶基因的成比例激活水平。在第三个目标中，我们将使用光学方法在群体和单细胞水平上研究最近描述的主孢子形成调节剂的脉动动力学的生物学意义。特别是，我们将评估我们最近的假设，即孢子形成脉冲必须发生在DNA复制后，以确保孢子继承染色体和一个普遍的假设，即必须达到超阈值浓度的主孢子形成调节剂的细胞致力于孢子形成。从B开始。枯草芽孢杆菌孢子形成和应激反应回路在医学上重要的孢子形成细菌（包括B）中是广泛保守的。蜡状芽孢杆菌（B. cereus）、B. anthracis和C.我们的研究结果不仅将使细胞决策的理解取得突破，而且还为设计新的抗菌剂提供了基础。

项目成果

An open-hardware platform for optogenetics and photobiology.

• DOI: 10.1038/srep35363
• 发表时间: 2016-11-02
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Gerhardt KP;Olson EJ;Castillo-Hair SM;Hartsough LA;Landry BP;Ekness F;Yokoo R;Gomez EJ;Ramakrishnan P;Suh J;Savage DF;Tabor JJ
• 通讯作者: Tabor JJ

An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range.

具有超过10000倍动态范围的工程枯草芽孢杆菌诱导启动子系统。

• DOI: 10.1021/acssynbio.8b00469
• 发表时间: 2019-07-19
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Castillo-Hair, Sebastian M.;Fujita, Masaya;Igoshin, Oleg A.;Tabor, Jeffrey J.
• 通讯作者: Tabor, Jeffrey J.

FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.

• DOI: 10.1021/acssynbio.5b00284
• 发表时间: 2016-07-15
• 期刊: ACS synthetic biology
• 影响因子: 4.7
• 作者: Castillo-Hair SM;Sexton JT;Landry BP;Olson EJ;Igoshin OA;Tabor JJ
• 通讯作者: Tabor JJ