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。随后，他们通过合成化合物，对化合物有了更全面的掌握和了解。生物学现在正在经历一个类似的过渡，从破译生物物种的DNA序列信息的时代到合成基因组时代;这种过渡需要一个全新的生物学理解水平，这已经被正式定义为“合成生物学”（SynBio）的新学科。本课题以真核生物S.酿酒酵母作为具有合成基因组“Sc2.0”的细胞的基础，可用于回答关于染色体的基本性质、基因组组织、基因内容、RNA剪接的功能、小RNA在真核生物学中发挥作用的程度、原核生物与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的问题。完全合成的基因组的可用性将允许直接测试其他方法无法解决的进化问题。S.酿酒酵母是这些研究的首选生物，因为可用的基因组和相关资源比任何其他生物都要好。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将不同于天然生物体，并且可用于生物体的多种遗传测定可用于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努力，开设了一门新的本科课程，“构建基因组”，学生们在该课程中制作用作染色体组装起始材料的积木。这门课程将通过“特许经营”来扩大到其他学院和大学，从而直接参与项目的高度积极性的劳动力，并为全国范围内的学生提供无与伦比的培训/学习机会，并最终在国际上。最终的“合成酵母”将被设计和提炼，很可能发挥重要的实际作用。酵母菌和S.特别是酿酒酵母是用于工业发酵的卓越生物，具有广泛的实际用途，包括从农产品和副产品生产乙醇。生物伦理和安全问题：因为S。尽管酿酒酵母已经被人类食用了数千年，但它被美国食品和药物管理局正式认定为“一般安全”（GRAS）。此外，由于其一般无毒的性质，酵母S。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生物体。伦理和安全问题对研究人员来说很重要。为了指导他们在生物伦理方面的考虑，并帮助教育学生在这些问题上，研究人员与训练有素的生物伦理学家密切合作，对新兴技术有浓厚的兴趣。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，确保安全和负责任地使用将开发的技术。关于当前的Sc2.0项目，以下安全实践已纳入研究计划。 为了防止合成酵母菌株的释放，实验室维持在生物安全2级。 在不太可能的情况下释放到野外，合成菌株将处于与野生型酵母的严重竞争劣势，因为它们都是营养缺陷型的（依赖于营养补充剂）。营养缺陷型突变是不能回复的缺失，并且使用的所有菌株都携带至少两个这样的突变。一旦完整的基因组合成完成，一个正交的tRNA/合成酶对可以用来使酵母依赖于合成氨基酸，有效地防止在自然环境中的任何生长。 其他内在的“死亡开关”设计也正在研究中。 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，遗传物质与野生型菌株的交换将是不可能的，从而增加了遗传隔离。 一次引入一小部分天然基因组-通常为1%或更少-允许监测任何表型变化。

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.