Synthesis And Restructuring of a Yeast Chromosome

酵母染色体的合成和重组

• 批准号: 1026068
• 负责人: Jef Boeke
• 金额: $ 219.25万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1026068&HistoricalAwards=false
• 关键词: Synthesis; Restructuring; Yeast; Chromosome

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.

化学家首先使用分析方法探索物质的结构，描述他们的感知。随后，他们通过合成化合物，对化合物有了更彻底的掌握和了解。生物学现在正在经历类似的转变，从破译生物物种DNA序列信息的时代到合成基因组时代；这种转变需要对生物学的理解达到一个全新的水平，这已被正式确定为“合成生物学”（SynBio）这一新学科。该项目使用真核生物酿酒酵母模型作为具有合成基因组“Sc2.0”的细胞的基础，该细胞可用于回答有关染色体基本特性、基因组组织、基因内容、功能的各种深刻问题。 RNA剪接的原理、小RNA在真核生物学中发挥作用的程度、原核生物和真核生物之间的区别，以及基因组结构和进化之间的密切关系。完全合成的基因组的可用性将允许直接测试其他方式无法解决的进化问题。酿酒酵母是这些研究的首选生物体，因为可用的基因组和相关资源比任何其他生物体都要好。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，并且可用于该生物体的多种遗传测定可用于了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在一门新的本科课程“构建基因组”上，学生可以在其中制作用作染色体组装起始材料的构建块。该课程将通过“特许经营”给其他学院和大学来大幅扩展，从而使积极主动的劳动力直接参与该项目，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”很可能发挥重要的实际作用。酵母，特别是酿酒酵母，是工业发酵的杰出生物体，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：由于酿酒酵母已被人类食用数千年，因此美国食品和药物管理局正式将其“普遍认为安全”(GRAS)。此外，由于其一般无毒的性质，酿酒酵母不受重组 DNA 咨询委员会的重组 DNA 监管。因此，它可以说是合成基因组学的最佳生物体。道德和安全问题对研究人员来说很重要。为了指导他们考虑生物伦理并帮助教育学生这些问题，研究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。该项目还包括公众参与，旨在解决与 SynBio 相关的合法社区担忧。研究人员将采取一切必要措施，确保安全和负责任地使用将要开发的技术。关于当前的 Sc2.0 项目，以下安全实践已纳入研究计划中。 为了防止合成酵母菌株的释放，实验室的生物安全等级保持在 2 级。万一释放到野外，合成菌株将在与野生型酵母的竞争中处于严重劣势，因为它们都是营养缺陷型的（依赖于营养补充剂）。营养缺陷型突变是无法恢复的缺失，所有使用的菌株都至少携带两个此类突变。一旦全基因组合成完成，正交 tRNA/合成酶对可用于使酵母依赖于合成氨基酸，从而有效防止在自然环境中的任何生长。 其他内在的“终止开关”设计也在研究中。 随着越来越多的合成片段积累以及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不太可能，从而增加遗传隔离。 一次引入一小部分天然基因组（通常为 1% 或更少），从而可以监测发生的任何表型变化。