Synthesis And Restructuring of a Yeast Chromosome

化学家首先使用分析方法探索物质的结构，描述他们所感知的东西。随后，他们通过合成化合物获得了更彻底的掌握和对化合物的洞察。生物学目前正经历着从破译生物物种DNA序列信息的时代到合成基因组时代的类似转变；这种转变要求对生物学的理解达到一个全新的水平，这已被正式化为合成生物学(SynBio)这一新学科。该项目使用模式真核生物酿酒酵母作为具有合成基因组“Sc2.0”的细胞的基础，该基因组可用于回答关于染色体的基本属性、基因组组织、基因内容、RNA剪接的功能、小RNA在真核生物学中的作用程度、原核生物和真核生物之间的区别以及基因组结构与进化之间的密切关系等各种深刻问题。全合成基因组的可获得性将允许对进化问题进行直接测试，否则这些问题是无法接近的。酿酒酵母是这些研究的首选生物体，因为现有的基因组和相关资源比其他任何生物体都要好。这为将广泛的酵母系统生物学信息应用于有机体的染色体设计提供了机会。毫无疑问，Sc2.0将不同于原生生物体，并且可用于该生物体的大量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：主要研究人员投入了大量的精力和精力，开设了一门新的本科课程--“构建基因组”，在这门课程中，学生们生产用作染色体组装起始材料的积木。这门课程将通过将其特许经营到其他学院和大学而大幅扩展，从而使高度积极的劳动力直接参与到该项目中，并为全国乃至国际学生提供无与伦比的培训/学习机会。最终将被设计和提炼的“合成酵母”可能会发挥重要的实际作用。酵母菌，特别是酿酒酵母，是工业发酵的优势生物，具有广泛的实际用途，包括从农产品和副产品中生产乙醇。生物伦理和安全问题：由于酿酒酵母几千年来一直被人类食用，美国食品和药物管理局(FDA)正式将其列为“一般安全”(GRAS)。此外，由于其一般无害的性质，酿酒酵母免除了重组DNA咨询委员会对重组DNA的监管。因此，它可以说是合成基因组学最好的有机体。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，并在这些问题上帮助教育学生，调查人员与一位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。该项目还包括旨在解决社区与SynBio相关的合理关切的公众参与。调查人员将采取一切必要步骤，确保安全和负责任地使用将开发的技术。关于即刻Sc2.0项目，以下安全实践被整合到研究计划中。为了防止合成酵母菌株的释放，实验室维持在生物安全级别2。如果不太可能释放到野外，合成菌株将处于与野生类型酵母的严重竞争劣势，因为它们都是营养缺陷型(依赖营养补充剂)。营养缺乏性突变是不能逆转的缺失，所有使用的菌株都至少携带两个这样的突变。一旦完成全基因组合成，就可以使用一对正交的tRNA/合成酶来使酵母依赖于合成的氨基酸，从而有效地防止在自然环境中的任何生长。其他固有的“终止开关”设计也在研究中。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，与野生型菌株的遗传物质交换将不太可能，从而增加遗传隔离。一次引入一小部分本地基因组--通常是1%或更少--以便在发生任何表型变化时进行监测。

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.