RII Track-4: Selection of methylxanthine-responsive aptamers

非技术性说明本项目旨在建设亚拉巴马大学塔斯卡卢萨分校（UA）在微生物工程日益增长领域的研究能力。凭借丰富的自然可再生资源，亚拉巴马是研究代谢工程、合成生物学和工业微生物学以生产生物基燃料和化学品的理想地点。然而，在这些领域的研究很少，这些领域对整个国家的科学界和工业部门越来越重要。该项目将允许来自UA的研究人员在耶鲁大学进行研究，学习新技术并创建新工具来设计微生物细胞。这项研究旨在构建新的系统，以控制特定微生物酶在分子水平上的生产，特别注意开发系统，控制参与咖啡因代谢的酶的生产。这些系统将在发现用于制药应用的新酶和开发新的诊断工具以检测咖啡因代谢方面有很大的好处。这项工作最初将为一名研究生提供培训，其结果将纳入本科课程和研究。通过这个项目形成的研究合作将有助于发展在UA进行代谢工程和合成生物学的新的和不断增长的领域的研究能力，提供额外的机会，教育和培训研究生和本科生。技术说明拟议的工作的目标是分离适体和构建核糖开关响应甲基黄嘌呤可可碱，副黄嘌呤和7-甲基黄嘌呤。将通过多轮体外选择文库来分离适体，所述文库由连接至锤头状核酶的茎II的40个核苷酸的随机片段组成。使用分离的最佳适体，将构建核糖开关以通过翻译抑制或mRNA稳定性机制控制基因表达，并将其置于编码绿色荧光蛋白的基因的上游以表征其体内活性。研究将在耶鲁大学的一个实验室进行，这些方法是在那里开创的。本项目中产生的核糖开关和适体将极大地有利于开发用于制药和化妆品行业的甲基黄嘌呤产生突变酶，从而能够检测细胞内咖啡因代谢物，并产生体外和体内检测咖啡因代谢的工具。此外，这项工作将提供一个模板，为建设的代谢途径，其中每一种酶是由一个核糖开关控制的酶？的底物，并将提高我们的知识合成核糖开关建设。在这里开发的合作将产生高质量的本科生和研究生的研究机会，同时大大扩大微生物工程和合成生物学的研究能力在UA。

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.