RII Track-4: Selection of methylxanthine-responsive aptamers

非技术描述该项目旨在为阿拉巴马大学塔斯卡卢萨分校(UA)不断增长的微生物工程领域的研究建立能力。阿拉巴马州拥有丰富的自然可再生资源，是代谢工程、合成生物学和生产生物燃料和化学品的工业微生物学研究的理想地点。然而，在这些领域的研究非常少，而这些领域对全州的科学界和工业部门越来越重要。该项目将允许来自加州大学的研究人员在耶鲁大学进行研究，学习新技术，并创造新的工具来设计微生物细胞。这项研究试图构建新的系统，在分子水平上控制特定微生物酶的产生，并特别关注开发控制咖啡因代谢相关酶的产生的系统。这些系统将在发现用于制药的新酶以及开发检测咖啡因代谢的新诊断工具方面大有裨益。这项工作最初将为一名研究生提供培训，结果将整合到本科课程和研究中。通过该项目形成的研究合作将有助于发展加州大学在代谢工程和合成生物学新的和不断增长的领域进行研究的能力，提供更多的机会来教育和培训研究生和本科生。技术说明拟议工作的目标是分离适体并构建对甲基黄嘌呤可可碱、对黄嘌呤和7-甲基黄嘌呤有反应的核糖开关。将通过多轮体外选择文库来分离适体，文库由连接在锤头状核酶茎II上的40个核苷酸随机片段组成。利用分离到的最好的适体，将构建核糖开关，通过翻译抑制或mRNA稳定机制来控制基因的表达，并将其置于编码绿色荧光蛋白的基因的上游，以表征其在体内的活性。研究将在耶鲁大学的一个实验室进行，这些方法就是在那里开创的。该项目中产生的核糖开关和适体将在开发用于制药和化妆品行业的甲基黄嘌呤产生突变酶方面大有裨益，能够检测细胞内的咖啡因代谢物，并产生体外和体内检测咖啡因代谢的工具。此外，这项工作将为构建代谢途径提供一个模板，在代谢途径中，每个酶由酶-S底物特有的核糖开关控制，并将提高我们对合成核糖开关结构的了解。在这里开展的合作将创造高质量的本科生和研究生研究机会，同时极大地扩展加州大学的微生物工程和合成生物学研究能力。

Non-technical DescriptionThis project seeks to build capacity for research in the growing field of microbial engineering at the University of Alabama at Tuscaloosa (UA). With a wealth of natural renewable resources, Alabama is an ideal location for research in metabolic engineering, synthetic biology, and industrial microbiology for the production of bio-based fuels and chemicals. However, there is very little research in these areas, which are of increasing importance to the scientific community and industrial sector throughout the state. This project will allow researchers from UA to conduct studies at Yale University, learning new techniques and creating new tools to engineer microbial cells. This research seeks to construct new systems to control the production of specific microbial enzymes at the molecular level, with particular attention to developing systems that control the production of enzymes involved in caffeine metabolism. These systems will be of great benefit in discovering new enzymes for pharmaceutical applications, and developing new diagnostic tools to detect caffeine metabolism. This work will initially provide training for one graduate student, and results will be integrated into undergraduate curriculum and research. The research collaboration formed through this project will help develop capacity at UA to perform research in the new and growing fields of metabolic engineering and synthetic biology, providing additional opportunities to educate and train graduate and undergraduate students.Technical DescriptionThe goal of the proposed work is to isolate aptamers and construct riboswitches responsive to the methylxanthines theobromine, paraxanthine, and 7-methylxanthine. Aptamers will be isolated through multiple rounds of in vitro selection of a library composed of a 40 nucleotide random fragment attached to stem II of the hammerhead ribozyme. Using the best aptamers isolated, riboswitches will be constructed to control gene expression through translation inhibition or mRNA stability mechanisms, and placed upstream of the gene encoding green fluorescent protein to characterize their activity in vivo. Research will be performed at a laboratory at Yale University, where these methods were pioneered. The riboswitches and aptamers generated in this project will be of great benefit in developing methylxanthine-producing mutant enzymes for use in the pharmaceutical and cosmetic industries, enabling detection of intracellular caffeine metabolites, and generating in vitro and in vivo tools to detect caffeine metabolism. Additionally, the work will provide a template for construction of metabolic pathways in which each enzyme is controlled by a riboswitch specific for the enzyme?s substrate and will improve our knowledge of synthetic riboswitch construction. The collaboration developed here will generate high quality undergraduate and graduate research opportunities while greatly expanding the microbial engineering and synthetic biology research capabilities at UA.