Synthesis And Restructuring of a Yeast Chromosome

Johns Hopkins University, Baltimore MD

Investigators

Abstract

Chemists first probed the structure of matter using analytic approaches, describing what they perceived. They subsequently gained a far more thorough mastery of and insights into chemical compounds by synthesizing them. Biology is now undergoing a similar transition from the age of deciphering DNA sequence information of biological species to a synthetic genome age; this transition demands a whole new level of biological understanding, which has been formalized as the new discipline of "Synthetic Biology" (SynBio). This project uses the model eukaryote S. cerevisiae as the basis for a cell with a synthetic genome "Sc2.0" that can be used to answer a wide variety of profound questions about fundamental properties of chromosomes, genome organization, gene content, the function of RNA splicing, the extent to which small RNAs play a role in eukaryotic biology, the distinction between prokaryotes and eukaryotes, and the intimate relationship between genome structure and evolution. The availability of a fully synthetic genome will allow direct testing of evolutionary questions that are not otherwise approachable. S. cerevisiae is the organism of choice for these studies because the genomic and related resources available are quite simply better than for any other organism. This offers the opportunity to apply extensive yeast systems biology information to the design of chromosomes for the organism. Undoubtedly, Sc2.0 will differ from the native organism, and the multitude of genetic assays available for the organism can be used to understand phenotypic differences that might be observed. Broader Impacts: A great deal of energy and effort has been invested by the principal investigators into a new undergraduate course, "Build A Genome", in which students produce the Building Blocks used as starting materials for chromosome assembly. This course will be expanded dramatically by "franchising" it to other Colleges and Universities, thereby engaging a highly motivated workforce directly in the project and providing unparalleled training/learning opportunities for students nationwide, and eventually, internationally. The eventual "synthetic yeast" that will be designed and refined is likely to play an important practical role. Yeasts, and S. cerevisiae in particular, are preeminent organisms for industrial fermentations, with a wide variety of practical uses including ethanol production from agricultural products and by-products. Bioethical and Safety Issues: Because S. cerevisiae has been consumed by humans for millennia, it is officially "Generally Regarded as Safe" (GRAS) by the U.S. Food and Drug Administration. Also, due to its generally innocuous nature, the yeast S. cerevisiae was exempted from recombinant DNA regulation by the Recombinant DNA Advisory Committee. It is therefore arguably the best organism for synthetic genomics. Ethical and safety matters are important to the investigators. To guide them in bioethical considerations and to help educate students in these matters, the investigators work closely with a trained bioethicist with strong interests in emerging technologies. The project also includes public engagement aimed at addressing legitimate community concerns associated with SynBio. The investigators will take all necessary steps to ensure the safe and responsible use of the technologies that will be developed. With regard to the immediate Sc2.0 project, the following safety practices are integrated into the research program. To guard against release of the synthetic yeast strains, the laboratory is maintained at Biosafety Level 2. In the unlikely event of release into the wild, the synthetic strains would be at a severe competitive disadvantage with wild-type yeast because they are all auxotrophic (dependent on nutritional supplements). The auxotrophic mutations are deletions that cannot be reverted, and all strains being used carry at least two such mutations. Once full genome synthesis is complete, an orthogonal tRNA/syntethase pair can be used to make the yeast dependent on a synthetic amino acid, effectively preventing any growth in a natural environment. Other intrinsic "kill switch" designs are also being investigated. Exchange of genetic material with wild type strains will be unlikely as more and more synthetic segments accumulate and as a planned chromosomal translocation is introduced, thus increasing genetic isolation. A small percentage of the native genome - typically 1% or less - is introduced at a time, allowing monitoring of any phenotypic changes as they occur.