BglBricks: A flexible standard for biological part assembly.
BglBricks: A flexible standard for biological part assembly.
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DOI：
10.1186/1754-1611-4-1
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发表时间：
2010-01-20
影响因子：
5.6
通讯作者：
Keasling JD
中科院分区：
文献类型：
--
作者：
Anderson JC;Dueber JE;Leguia M;Wu GC;Goler JA;Arkin AP;Keasling JD
标准生物部件，如BioBricks™部件，为新的工程学科提供了基础，使合成生物系统的设计和构建在生物能源，新材料，治疗和环境修复方面具有各种应用。虽然最初的BioBricks™组装标准已经被广泛使用，但它有几个缺点限制了其潜在应用范围。特别是，由于编码帧内停止密码子的不利疤痕序列，该系统不适合构建蛋白质融合体。在这里，我们提出了一个类似但新的组合标准，称为BglBricks，它解决了与原始标准相关的疤痕翻译问题。新系统采用BglII和BamHI限制性内切酶，这两种具有广泛使用历史的强大切割器，可产生编码甘氨酸-丝氨酸的6核苷酸疤痕序列，甘氨酸-丝氨酸是大多数蛋白质融合应用中无害的肽连接物。我们展示了新标准在三种不同应用中的实用性，包括构建具有广泛表达谱的组成活性基因表达装置，构建嵌合，多结构域蛋白融合，以及将功能DNA序列靶向整合到大肠杆菌基因组的特定位点。BglBrick标准提供了一个新的、更灵活的平台，用于生成标准生物部件和自动DNA组装。BglBrick组装反应以及自动化和生物信息学工具的开发工作目前正在进行中。这些工具将为将基因工程从技术密集型艺术转变为纯粹基于设计的学科提供基础。
Standard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon. Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome. The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.