The Assemblatron

项目摘要 快速生成合成核酸结构的方法已经极大地改变了生物学和 生物医学研究。这些领域的改进将继续影响基因组学的各个领域， 生物医学，如合成基因组学和相关的功能筛选。实现显著的进步， 新的核酸合成和合成构造能力有可能导致显著的 提高对疾病的了解、诊断、治疗和预防；在农业方面的进展， 环境科学和补救；以及我们对进化和生态系统的理解。 目前使用各种合成结构的能力是通过降低成本实现的 寡核苷酸合成以及用于更大结构的分级组装的极大改进的技术， 主要是在酵母中。我们的团队在组装酵母菌基因组(即国际Sc2.0)方面处于领先地位 项目)，并在NHGRI赞助的CEGS中启动了“暗物质项目”，目的是在功能上 剖析非编码DNA及其对人类/哺乳动物转录调控的贡献。我们还有 开发了一个链接到我们的LIMS的内部设计软件应用程序环境，名为Mendel(指导 设计环境和LIMS)。但我们需要做更多的工作，使这些项目变得更加容易完成- 最终，在任何实验室。 在这个项目中，我们计划开发“ASSEBLATRON”，一个工作流/设计平台/主机向量系统， 系统地优化了“酵母组装”过程，并能够生产非常大的DNA分子 最大可达一兆数据库。我们将开发一个人可以组装1-2兆数据库的系统 在几天的工作中获得20-30kb的DNA片段，在2-4周内获得1-2Mb的~100kb的DNA片段。具体目标 IS&gt；将大DNA组装效率提高10倍，表现为单个研究人员的能力，从 使用3kb的起始材料，完成以下操作：1)在1-2周内组装1-2Mb的DNA，2)完成 在2-4周内组装成10到20个100+kb的片段。最终，这项研究计划将导致发展 指将大部分或全部过程自动化的装配线装置。我们计划在以下方面实现这一改进 效率，这目前受到一系列瓶颈的限制，这些瓶颈存在于使用 干法和湿法相结合的实验室方法，概述如下。

Project Summary Methods for rapidly generating synthetic nucleic acid constructs have dramatically changed biological and biomedical research. Improvements in these arenas will continue to impact varied areas of genomics and biomedicine such as synthetic genomics and associated functional screens. Enabling significant advances with new nucleic acid synthesis and synthetic construct capabilities has the potential to lead to remarkable improvements in the understanding, diagnosis, treatment and prevention of disease; advances in agriculture, environmental science and remediation; and our understanding of evolution and ecological systems. Current abilities to work with a variety of synthetic constructs have been enabled by cost reductions in oligonucleotide synthesis along with vastly improved techniques for hierarchal assembly of larger constructs, largely in yeast. Our group has led the way both in assembling the yeast genome (i.e. the international Sc2.0 project) and in a NHGRI-sponsored CEGS that has launched the “Dark Matter Project”, aiming to functionally dissect noncoding DNA and its contribution to human/mammalian transcriptional regulation. We have also developed an in-house design software application environment linked to our LIMS called MenDEL (Mentored Design Environment and LIMS). But we need to do much more to make such projects ever easier to do – ultimately, in any lab. In this project we plan to develop the “Assemblatron”, a workflow/Design platform/Host vector system that systematically optimizes the “Yeast assembly” process, and is capable of producing very large DNA molecules of up to a megabase in size. We will develop a system in which one person can assemble 1-2 Megabases of 20-30 kb DNA pieces in a few days’ work, and 1-2 Mb of ~100 kb pieces in 2-4 weeks. The specific goal is >10X improvement in Big DNA assembly efficiency, manifested as the ability of a single researcher, starting with 3 kb starting materials, to do the following: 1) Assemble 1-2 Mb of DNA in 1-2 weeks and 2) Finalize assembly into 10 to 20 100+ kb pieces in 2-4 weeks. Ultimately this research program will lead to development of an Assemblatron device that automates much or all of the process. We plan to achieve this improvement in efficiency, which is currently limited by a series of bottlenecks that exist throughout the workflow using a combination of dry lab and wet lab methods, outlined below.