Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
J Biol Eng. 2010 Jan 20;4(1):1. doi: 10.1186/1754-1611-4-1.
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.
标准生物部件，如 BioBricks 部件，为一个新的工程学科提供了基础，使设计和构建具有各种应用的合成生物系统成为可能，这些应用包括生物能源、新材料、治疗学和环境修复。尽管原始的 BioBricks 组装标准得到了广泛的应用，但它有几个缺点，限制了其潜在应用的范围。特别是，由于编码一个无义终止密码子的不利疤痕序列，该系统不适合构建蛋白质融合。
在这里，我们提出了一个类似但新的组成标准，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 限制酶，这两种酶是用途广泛的强有力的切割酶，产生的 6 个核苷酸疤痕序列编码甘氨酸-丝氨酸，这是大多数蛋白质融合应用中无毒性的肽接头。我们在三个不同的应用中展示了新标准的实用性，包括构建具有广泛表达谱的组成型激活基因表达装置，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基因座。
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化 DNA 组装。目前正在进行 BglBrick 组装反应的研究，以及自动化和生物信息学工具的开发。这些工具将为将基因工程从一项技术密集型艺术转变为一项纯粹基于设计的学科提供基础。
