CAREER: Riboswitch-based whole-cell biosensors to detect small organic molecules: A combined educational and research plan

职业：基于核糖开关的全细胞生物传感器检测有机小分子：教育和研究相结合的计划

• 批准号: 1148202
• 负责人: Jane Liu
• 金额: $ 40.48万
• 依托单位: Drew University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-15 至 2012-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1148202&HistoricalAwards=false
• 关键词: CAREER; Riboswitch; based; whole; cell

1148202LiuDrew UniversityAlthough small organic molecules are essential to all biological systems, there remains a need for tools that allow scientists to measure the concentration of any given small molecule in a biologically relevant setting. The project leader's long-term research goal is to develop a general method to rapidly and easily monitor small molecules relevant to the study of biology and the environment. The objective of this proposal is to engineer riboswitch-based biosensors to monitor intracellular concentrations of small organic molecules. Riboswitches are naturally occurring non-coding RNA elements that act as direct sensors of diverse small molecules and can signal molecular recognition through altered protein expression. The rationale for this project is that the demonstrated plasticity of riboswitches coupled with the utility of genetic selections can form the basis of a powerful method for engineering whole-cell biosensors for small molecules. The proposed work sets out to demonstrate the utility of riboswitch-based biosensors by using a naturally occurring riboswitch to identify environmental factors that may contribute to the metabolism of an important signaling molecule, cyclic diguanylate, in Vibrio cholerae. To demonstrate how genetic selections can modify and improve natural riboswitches for use in biosensors, the V. cholerae-specific cyclic diguanylate riboswitch will also be re- engineered to generate a new riboswitch that can monitor cyclic diguanylate in any bacterial species. The potential of such a tool is immense in terms of its ability to advance the entire field of microbiology research. Finally, to demonstrate the full range and potential of this approach, a riboswitch-based biosensor for polychlorinated biphenyls will be engineered through directed evolution. The successful completion of this aim would produce a biosensor that can provide a sensitive and inexpensive means to monitor bioavailable and bioaccessible levels of a persistent organic pollutant. These models will define the precision with which riboswitch-based biosensors can be generated and will speak to their broad utility. This contribution is transformative because it is expected to provide a system that will be able to rapidly detect and report the intracellular level of virtually any cell permeant small molecule of interest. The engineering of biosensors and the use of non-coding RNAs as research tools, moreover, present an exciting frontier for a diverse group of budding scientists. The project leader is committed to graduating scientifically literate undergraduates and motivating students to pursue life-long careers in science; this project directly addresses these career goals. Over a dozen undergraduate summer research positions will be funded over the five-year duration of the project. Students working on this project will communicate their results to the scientific community as co-authors of journal articles and as presenters at professional meetings. Student researchers will also be asked to contribute to a blog which seeks to communicate to other undergraduate and high school students what science research is and what it produces. As part of this project, authentic research experiences will be introduced into the curriculum for both majors and non-majors. In addition to providing experiential learning on-campus, the project leader is also initiating a partnership with a research-intensive institution to create off-campus research opportunities for her undergraduate students.

1148202刘德鲁大学尽管有机小分子对所有生物系统都是必不可少的，但仍然需要一种工具，使科学家能够在生物学相关环境中测量任何给定小分子的浓度。该项目负责人的长期研究目标是开发一种通用方法，以快速简便地监测与生物学和环境研究相关的小分子。本提案的目的是设计基于核糖开关的生物传感器来监测细胞内小有机分子的浓度。核糖开关是天然存在的非编码RNA元件，其充当不同小分子的直接传感器，并且可以通过改变蛋白质表达来发出分子识别信号。该项目的基本原理是，核糖开关的可塑性与遗传选择的效用相结合，可以形成一个强大的方法的基础上工程全细胞生物传感器的小分子。拟议的工作阐述了利用核糖开关为基础的生物传感器的效用，通过使用天然存在的核糖开关，以确定环境因素，可能有助于代谢的一个重要的信号分子，环二鸟苷酸，霍乱弧菌。为了证明遗传选择如何修饰和改善用于生物传感器的天然核糖开关，霍乱弧菌特异性环状二鸟苷酸核糖开关也将被重新工程化以产生可以监测任何细菌物种中的环状二鸟苷酸的新核糖开关。这种工具的潜力是巨大的，因为它能够推动整个微生物学研究领域的发展。最后，为了展示这种方法的全部范围和潜力，将通过定向进化设计基于核糖开关的多氯联苯生物传感器。这一目标的成功实现将产生一种生物传感器，它可以提供一种灵敏和廉价的手段来监测持久性有机污染物的生物可利用和生物可获得水平。这些模型将定义基于核糖开关的生物传感器可以产生的精度，并将说明其广泛的实用性。这一贡献是变革性的，因为它预计将提供一种系统，该系统将能够快速检测和报告几乎任何感兴趣的细胞渗透性小分子的细胞内水平。此外，生物传感器的工程设计和使用非编码RNA作为研究工具，为不同的新兴科学家提供了一个令人兴奋的前沿。项目负责人致力于毕业的科学素养的本科生和激励学生追求终身的科学事业;这个项目直接解决这些职业目标。 在该项目的五年期间，将资助十几个本科生暑期研究职位。从事该项目的学生将作为期刊文章的共同作者和专业会议的演讲者向科学界传达他们的结果。学生研究人员还将被要求贡献一个博客，旨在传达给其他本科生和高中学生什么是科学研究，它产生什么。作为该项目的一部分，真实的研究经验将被引入专业和非专业的课程。除了提供校内体验式学习外，项目负责人还与一家研究密集型机构建立了合作伙伴关系，为她的本科生创造校外研究机会。