j5 DNA Assembly Design Automation Software
j5 DNA Assembly Design Automation Software
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DOI：
10.1021/sb2000116
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发表时间：
2012-01-01
影响因子：
4.7
通讯作者：
Keasling, Jay D.
中科院分区：
文献类型：
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作者：
Hillson, Nathan J.;Rosengarten, Rafael D.;Keasling, Jay D.
合成生物学的最新进展已经产生了标准化和自动化的DNA组装协议，使广泛的生物技术研究和开发成为可能。不幸的是，现代无疤痕多部分DNA组装方法所需的实验设计通常是费力、耗时和容易出错的。在这里，我们报告了基于web的软件工具j5的开发和部署，该工具可以自动设计无疤痕多部分DNA组装协议，包括SLIC， Gibson， CPEC和Golden Gate。j5设计过程的关键创新包括成本优化、在成本有效的情况下利用DNA合成、设计规范规则的实施、分层组装策略以减轻可能的组装错误，以及手动或自动构建无疤痕组合DNA文库的指导。使用GFP表达测试平台，我们证明j5设计可以用SLIC， Gibson或CPEC组装方法执行，用于用金门组装方法构建组合文库，并应用于大肠杆菌线性基因缺失盒的制备。本文报道的DNA组装设计算法一般适用于广泛的DNA构建方法，并可用于补充其他DNA组装设计工具。总之，这些创新节省了研究人员的时间和精力，减少了用户设计错误和脱靶组装产品的频率，降低了研究成本，并使无疤痕的多部分和组合DNA构建在没有计算机辅助设计的情况下不可实现。
Recent advances in Synthetic Biology have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnological research and development. Unfortunately, the experimental design required for modem scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the preparation of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scarless multipart and combinatorial DNA construction at scales unfeasible without computer-aided design.