Establishing a generalizable model for predictable antisense RNA repression

RNA主要被认为是传递遗传信息的信使，而小RNA在基因调控中起着重要作用。已经发现这种调控RNA天然存在于许多生物体中，但是它们已经在相对有限数量的细菌中用作调控基因表达的工具。在这个项目中，研究人员将开发可推广的RNA调节剂，可用于理解和工程化不同的生物技术重要的细菌。具体而言，反义RNA（asRNA），在细菌中的常见的调节RNA之一，设计原则将被确定为生物学家和生物工程师提供可预测的RNA工具的基因调控。此外，该项目通过培养下一代产生了广泛的影响。多名研究生将接受培训，以执行跨学科项目。此外，我们的拓展计划还将向高中教师介绍最先进的合成生物学RNA工具，并为他们提供用于课堂的教学工具包，扩大学生对科学和工程的参与。本项目的长期目标是确定asRNA的定量设计原则，并提供一个可推广的模型，使可预测的可调asRNA介导的基因抑制在不同的生物体中。asRNA是几种类型的小调控RNA之一，它通过与其靶信使RNA结合来抑制基因表达。虽然这种结合遵循简单的RNA碱基配对规则，但可预测的可调asRNA介导的抑制仍然具有挑战性。通过在质粒中使用三个靶报告基因，确定了大肠杆菌DH10B中asRNA最重要的设计参数。将开发asRNA介导的阻遏的预测模型，该模型通过多元线性回归分析将阻遏效率与所鉴定的参数相关联。为了证明这个模型的通用性，提出了三个具体的目标。首先，在大肠杆菌中建立了质粒报告基因的模型。coli DH10B中的重组质粒将通过将其扩展到染色体基因靶点进行验证。这项任务将允许在不同的遗传背景下测试正向设计的asRNA。其次，预测模型将在三种不同的细菌物种中进行测试。这项任务将检验asRNA设计原则在不同细菌中是一致的这一假设。第三，该模型将通过构建和测试额外的asRNA来进一步验证。还将开发该模型的用户友好的在线版本，以提供一种合作推进这种基于RNA的技术的方法。

RNA is mostly known as a messenger to convey genetic information, while small RNA plays important roles in gene regulation. Such regulatory RNAs have been found to exist naturally in many organisms, but they have been used in a relatively limited number of bacteria as a tool to regulate gene expression. In this project, the investigators will develop generalizable RNA regulators that can be used to understand and engineer diverse biotechnologically important bacteria. Specifically, design principles for antisense RNA (asRNA), one of the common regulatory RNAs in bacteria, will be determined to provide biologists and bioengineers with predictable RNA tools for gene regulation. Additionally, this project is broadly impactful by cultivating the next generation. Multiple graduate students will be trained to perform the interdisciplinary project. Moreover, our outreach program will introduce state-of-the-art synthetic biology RNA tools to high school teachers, and provide them with teaching kits that will be used in their classroom, broadening student participation in science and engineering.The long-term goal of this project is to determine quantitative design principles for asRNAs and provide a generalizable model that enables predictably tunable asRNA-mediated gene repression in diverse organisms. asRNA is one of several types of small regulatory RNAs, and it represses gene expression by binding to its target messenger RNA. Although this binding occurs following the simple RNA base-pairing rule, predictably tunable asRNA-mediated repression is still challenging. The most important design parameters for asRNA in Escherichia coli DH10B have been identified by using three target reporter genes in plasmids. A predictive model for asRNA-mediated repression, which relates repression efficiency to the identified parameters by a multiple linear regression analysis, will be developed. To demonstrate the generalizability of this model, three specific aims are proposed. First, the model developed with plasmid reporters in E. coli DH10B will be validated by expanding it to chromosomal gene targets. This task will allow for testing of forward-designed asRNAs in different genetic contexts. Second, the predictive model will be tested in three different bacterial species. This task will test the hypothesis that asRNA design principles are consistent among diverse bacteria. Third, the model will be further validated by building and testing additional asRNAs. A user-friendly online version of the model will also be developed to provide a way to collaboratively advance this RNA-based technology.