Synthesis And Restructuring of a Yeast Chromosome

化学家首先用分析方法探索物质的结构，描述他们所感知到的东西。随后，他们通过合成化合物，对化合物有了更彻底的掌握和更深入的了解。生物学现在正经历着一个类似的转变，从破译生物物种DNA序列信息的时代到合成基因组时代；这种转变要求对生物学的理解达到一个全新的水平，这已经被正式定义为“合成生物学”（SynBio）的新学科。本项目以酿酒酵母模型真核生物为基础，构建具有“Sc2.0”合成基因组的细胞，该合成基因组可用于回答关于染色体基本特性、基因组组织、基因含量、RNA剪接功能、小RNA在真核生物生物学中的作用程度、原核生物与真核生物的区别以及基因组结构与进化之间的密切关系等一系列深刻问题。一个完全合成的基因组的可用性将允许直接测试进化问题，否则无法接近。酿酒葡萄球菌是这些研究的首选生物，因为它的基因组和相关资源比其他任何生物都要好。这提供了将广泛的酵母系统生物学信息应用于生物体染色体设计的机会。毫无疑问，Sc2.0将会不同于原生生物，并且大量可用的遗传分析可以用来理解可能观察到的表型差异。更广泛的影响：主要研究人员已经投入了大量的精力和努力来开设一门新的本科课程，“构建基因组”，在这门课程中，学生们制作用于染色体组装的初始材料的积木。这门课程将通过“特许经营”的方式扩展到其他学院和大学，从而直接参与到项目中来，并为全国乃至全球的学生提供无与伦比的培训/学习机会。最终设计和提炼的“合成酵母”可能会发挥重要的实际作用。酵母，特别是酿酒酵母，是工业发酵的卓越生物，具有各种各样的实际用途，包括从农产品和副产品中生产乙醇。生物伦理和安全问题：因为酿酒葡萄球菌已经被人类食用了几千年，它被美国食品和药物管理局正式认定为“一般认为安全”（GRAS）。此外，由于其一般无害的性质，酿酒酵母被重组DNA咨询委员会豁免重组DNA监管。因此，它可以说是合成基因组学的最佳有机体。道德和安全问题对调查人员来说很重要。为了指导他们在生物伦理方面的考虑，并帮助在这些问题上教育学生，研究人员与对新兴技术有浓厚兴趣的训练有素的生物伦理学家密切合作。该项目还包括公众参与，旨在解决与SynBio相关的合法社区问题。调查人员将采取一切必要措施，确保安全、负责任地使用即将开发的技术。关于当前的Sc2.0项目，以下安全实践被纳入研究计划。为防止合成酵母菌株释放，实验室维持在生物安全二级。在不太可能释放到野外的情况下，合成菌株与野生型酵母相比将处于严重的竞争劣势，因为它们都是营养不良的（依赖于营养补充剂）。营养不良突变是不能恢复的缺失，所有被使用的菌株都携带至少两种这样的突变。一旦全基因组合成完成，可以使用正交tRNA/合成酶对使酵母依赖于合成的氨基酸，有效地防止在自然环境中生长。其他内在的“死亡开关”设计也在调查中。随着越来越多的合成片段的积累和有计划的染色体易位的引入，从而增加了遗传隔离，与野生型菌株的遗传物质交换将不太可能。每次只引入一小部分原生基因组——通常是1%或更少——以便监测任何表型变化的发生。

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.