Automated Robotic Liquid Handling Assembly of Modular DNA Devices.
Automated Robotic Liquid Handling Assembly of Modular DNA Devices.
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DOI：
10.3791/54703
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发表时间：
2017-12-01
期刊：
影响因子：
--
通讯作者：
Densmore DM
中科院分区：
文献类型：
--
作者：
Ortiz L;Pavan M;McCarthy L;Timmons J;Densmore DM
模块化DNA组装技术的最新进展使合成生物学家能够测试更多可用的“设计空间”，这些“设计空间”是由由单个遗传成分组合而成的“装置”所代表的。然而，如此大量的设备的手动组装是耗时、容易出错且成本高昂的。合成生物学研究的日益复杂和规模要求有一种高效、可重复的方法来适应大规模、复杂和高通量的设备建设。在这里，使用基于类型IIS限制性内切酶的模块克隆(MoClo)技术的DNA组装协议在两个液体处理机器人平台上自动化。自动化液体处理机器人需要对不同粘度的液体(如酶、DNA、水、缓冲液)的移液参数进行仔细的、往往是繁琐的优化，以及显式编程，以确保正确地提取和分配DNA部件和试剂。这使得复杂程序集的手动脚本编写与手动DNA组装一样存在问题，并且需要一个能够自动生成脚本的软件工具。为此，我们开发了一个基于网络的软件工具http://mocloassembly.com，，用于从作为Genbank文件上传的基本DNA部分生成组合DNA设备库。我们提供对该工具的访问，以及我们的液体处理软件的导出文件，其中包括优化的液体类别、实验室设备参数和甲板布局。所有使用的DNA部件都可以通过Addgene获得，他们的数字地图可以通过波士顿大学BDC ICE注册中心访问。这些要素共同为其他组织自动化模块克隆实验和类似协议奠定了基础。这里介绍的自动化DNA组装工作流程能够实现DNA设备的可重复、自动化、高通量生产，并降低重复手动移液引起的人为错误的风险。测序数据显示，通过该工作流程产生的自动化DNA组装反应的准确率约为95%，与手动反应准备相比，所需动手时间仅为4%。
Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.
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影响因子：
4.2
作者：
Ma AC;McNulty MS;Poshusta TL;Campbell JM;Martínez-Gálvez G;Argue DP;Lee HB;Urban MD;Bullard CE;Blackburn PR;Man TK;Clark KJ;Ekker SC
通讯作者：
Ekker SC
影响因子：
46.9
作者：
Fussenegger, M;Morris, RP;Bailey, JE
通讯作者：
Bailey, JE
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48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
64.8
作者：
Ro, DK;Paradise, EM;Keasling, JD
通讯作者：
Keasling, JD
影响因子：
4.7
作者：
Bhatia, Swapnil;Densmore, Douglas
通讯作者：
Densmore, Douglas