EAGER: Collaborative Research: Integrating microtome sectioning with isotopic tracing to study biotransformation in synthetic Escherichia coli biofilms

EAGER：合作研究：将切片机切片与同位素示踪相结合，研究合成大肠杆菌生物膜的生物转化

• 批准号: 1700881
• 负责人: Yinjie Tang
• 金额: $ 3.25万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1700881&HistoricalAwards=false
• 关键词: EAGER; Collaborative; Research; Integrating; microtome

1700881/1700935Tang/RenBiofilms are created by colonies of bacteria with polymeric substances produced by the cells. With a high-level of tolerance to a variety of stresses, biofilms cause serious problems in both industrial (biofouling and corrosion) and healthcare (persistent infections) settings. On the other hand, biofilms of environmentally friendly bacteria have promising applications in bioremediation and biofuel production. This project will develop a new method for investigating nutrient transport and enzyme activity of using Escherichia coli as a model organism.The PIs have extensive research experience in E. coli metabolic analysis and biofilm physiology. This team will develop and test a new biofilm study method by integrating several interdisciplinary approaches: 1) culture synthetic biofilms with controlled morphology to reduce spatial variation in gene expression and enzymatic functions; 2) use microtome sectioning to obtain thin slides of biofilm samples with cells in similar metabolic/nutrient transport status; and, 3) apply a stable isotopic labeling method (i.e., 13C-pulse) to trace nutrient mass transfer within biofilm layers as well as intracellular free metabolite conversions along functional pathways. The transient labeling in cascade metabolites can reveal the speed of substrate diffusion through biofilm layers as well as the rate of biotransformation into downstream intracellular metabolites. It can also determine these active biotransformation pathways important for biofilm survival and growth. Ultimately, the outcome of this exploratory project will be a new method for studying biofilm physiology (i.e., intracellular biotransformation) of diverse environmental microbes. This biofilm study technology will reveal regulatory mechanisms of biofilm growth and persistence under environmental stresses (e.g., antibiotic conditions); and it will improve our understandings of diverse biofilm systems from environmental microbes to pathogens. Specifically, Escherichia coli will be used as a model bacterium in this project with complementary studies outlined in the following Tasks: 1. Create synthetic biofilms with rigorously controlled morphology to reduce structural heterogeneity. 2. Optimize microtome technology to quench and sample free metabolites from different layers of biofilm cells, which can be analyzed via liquid chromatography-mass spectrometry. 3. Validate this new method by conducting 13C-pulse experiments with microtome sectioning and liquid chromatography-mass spectrometry analysis of cascade metabolites to determine glucose transfer and biotransformation rates across different layers of E. coli biofilm cells. This project will result in a new approach to section biofilms to detect metabolite biotransformation and probe the pathway function with desired spatial resolutions. The interdisciplinary nature of this work and the development of new tools outlined herein will build the foundation for advanced research and innovation to address many grand challenges associated with microbial biofilms at molecular level. It will provide new insights into nutrient transport and intracellular enzyme conversions within the biofilm matrix; and help reveal regulatory mechanisms and essential pathways for biofilm survival under diverse environmental conditions. Such insights can help researchers design more effective way to control biofilm physiologies.

1700881/1700935TANG/肾脏是由细菌的菌落与细胞产生的聚合物物质产生的。具有对各种压力的高度耐受性，生物膜在工业（生物污染和腐蚀）和医疗保健（持续感染）环境中都会引起严重的问题。另一方面，环保细菌的生物膜在生物修复和生物燃料生产中具有有希望的应用。该项目将开发一种新方法，用于研究使用大肠杆菌作为模型生物的营养运输和酶活性。PI在大肠杆菌代谢分析和生物膜生理学方面具有丰富的研究经验。该团队将通过整合几种跨学科方法来开发和测试一种新的生物膜研究方法：1）具有控制形态的培养合成生物膜，以减少基因表达和酶促功能的空间变化； 2）使用微型截面获得具有类似代谢/养分转运状态的细胞的生物膜样品的薄玻片； 3）应用稳定的同位素标记方法（即13c-pulse）来追踪生物膜层中的养分传质，以及沿功能途径的细胞内游离代谢物转换。级联代谢产物中的瞬时标记可以揭示底物扩散的速度通过生物膜层，以及生物转化速率向下游细胞内代谢物。它还可以确定这些对生物膜存活和生长重要的活性生物转化途径。最终，该探索性项目的结果将是一种研究多种环境微生物的生物膜生理学（即细胞内生物转化）的新方法。这项生物膜研究技术将揭示在环境应力下（例如抗生素条件）在环境应力下生长和持久性的调节机制。它将提高我们对从环境微生物到病原体的各种生物膜系统的理解。具体而言，大肠杆菌将在该项目中用作模型细菌，其中包括以下任务中概述的互补研究：1。创建具有严格控制形态的合成生物膜，以降低结构异质性。 2。优化微型技术以从不同层的生物膜细胞淬灭和采样游离代谢物，可以通过液相色谱 - 质谱法对其进行分析。 3。通过对级联代谢产物的微型截面和液相色谱 - 质谱分析进行13C脉冲实验来验证这种新方法，以确定跨大肠杆菌生物膜细胞的葡萄糖转移和生物转化速率。该项目将导致一种新方法来检测代谢物生物转化并使用所需的空间分辨率探测途径功能。这项工作的跨学科性质以及本文概述的新工具的开发将为高级研究和创新建立基础，以应对分子水平上与微生物生物膜相关的许多巨大挑战。它将为生物膜基质内的营养运输和细胞内酶转化提供新的见解。并有助于揭示在各种环境条件下生物膜生存的调节机制和基本途径。这些见解可以帮助研究人员设计更有效的方法来控制生物膜生理。