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

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，但仍然需要工具来允许科学家在生物相关的环境中测量任何给定的小分子的浓度。项目负责人的长期研究目标是开发一种通用方法，快速、轻松地监测与生物学和环境研究相关的小分子。这项提议的目标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，可以作为各种小分子的直接传感器，并可以通过改变蛋白质表达来发出分子识别的信号。这个项目的基本原理是，核糖开关的可塑性，加上遗传选择的实用性，可以形成一种强大的方法来设计用于小分子的全细胞生物传感器。这项拟议的工作旨在通过使用自然产生的核糖开关来识别可能有助于霍乱弧菌中重要信号分子环二鸟酸盐代谢的环境因素，从而展示基于核糖开关的生物传感器的实用性。为了展示遗传选择如何修改和改进用于生物传感器的天然核糖开关，霍乱弧菌特有的环状二鸟苷核糖开关也将被重新设计，以产生一种新的核糖开关，可以监测任何细菌物种中的环二鸟苷酸。就推动整个微生物学研究领域的能力而言，这种工具的潜力是巨大的。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出一种基于核糖开关的多氯联苯生物传感器。这一目标的成功完成将产生一种生物传感器，它可以提供灵敏和廉价的手段来监测持久性有机污染物的生物可获得性和生物可获得性水平。这些模型将定义产生基于核糖开关的生物传感器的精确度，并将说明它们的广泛用途。这一贡献是变革性的，因为它有望提供一个系统，能够快速检测和报告几乎任何细胞内的水平，这意味着感兴趣的小分子。此外，生物传感器的工程和非编码RNA作为研究工具的使用，为不同的初出茅庐的科学家群体提供了一个令人兴奋的前沿。该项目负责人致力于让有科学素养的本科生毕业，并激励学生在科学领域追求终身职业生涯；该项目直接针对这些职业目标。在该项目为期五年的时间里，将资助十几个本科生暑期研究职位。从事这个项目的学生将作为期刊文章的合著者和专业会议的演讲者向科学界交流他们的结果。学生研究人员还将被要求建立一个博客，试图向其他本科生和高中生传播什么是科学研究以及它产生了什么。作为该项目的一部分，真实的研究经验将被引入到专业和非专业的课程中。除了在校园内提供体验式学习外，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。

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.