The Assemblatron

项目摘要 用于快速产生合成核酸构建体的方法已经显著改变了生物学和生物学特性， 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。实现重大进展， 新的核酸合成和合成构建体能力有可能导致显着的 提高对疾病的认识、诊断、治疗和预防;农业的进步， 环境科学和补救;以及我们对进化和生态系统的理解。 目前与各种合成构建体一起工作的能力已经通过以下方式实现： 寡核苷酸合成沿着用于较大构建体的分级组装的极大改进的技术， 主要是酵母。我们的团队在组装酵母基因组（即国际Sc2.0 项目）和NHGRI赞助的CEGS，该项目已经启动了“暗物质项目”，旨在从功能上 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 我开发了一个内部设计软件应用环境，与我们的LIMS连接，称为MenDEL（Mentored 设计环境和LIMS）。但我们需要做更多的工作，使这些项目更容易做- 最终，在任何实验室。 在这个项目中，我们计划开发“Assemblatron”，一个工作流程/设计平台/主机矢量系统， 系统地优化了“酵母组装”过程，并能够产生非常大的DNA分子 最大可达百万立方英尺。我们将开发一个系统，其中一个人可以组装1-2兆 20-30 kb的DNA片段在几天内完成，1-2 Mb的~100 kb的DNA片段在2-4周内完成。具体目标 大DNA组装效率提高10倍，表现为单个研究人员的能力， 1）在1-2周内组装1-2 Mb的DNA，2）完成 在2-4周内组装成10至20个100+ kb片段。最终，这项研究计划将导致发展 自动化大部分或全部过程的Assemblatron设备。我们计划在 效率，目前受到整个工作流程中存在的一系列瓶颈的限制， 干实验室和湿实验室方法的组合，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.