RII Track-4: Selection of methylxanthine-responsive aptamers

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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.