Leguia Mariana, Brophy Jennifer, Densmore Douglas, Anderson J Christopher
Department of Bioengineering, University of California, Berkeley, California, USA.
Methods Enzymol. 2011;498:363-97. doi: 10.1016/B978-0-12-385120-8.00016-4.
The primary bottleneck in synthetic biology research today is the construction of physical DNAs, a process that is often expensive, time-consuming, and riddled with cloning difficulties associated with the uniqueness of each DNA sequence. We have developed a series of biological and computational tools that lower existing barriers to automation and scaling to enable affordable, fast, and accurate construction of large DNA sets. Here we provide detailed protocols for high-throughput, automated assembly of BglBrick standard biological parts using iterative 2ab reactions. We have implemented these protocols on a minimal hardware platform consisting of a Biomek 3000 liquid handling robot, a benchtop centrifuge and a plate thermocycler, with additional support from a software tool called AssemblyManager. This methodology enables parallel assembly of several hundred large error-free DNAs with a 96+% success rate.
当今合成生物学研究的主要瓶颈在于物理DNA的构建，这一过程通常成本高昂、耗时且充满与每个DNA序列独特性相关的克隆难题。我们开发了一系列生物学和计算工具，降低了现有自动化和规模化的障碍，以实现大型DNA集的经济、快速且准确构建。在此，我们提供了使用迭代2ab反应对BglBrick标准生物部件进行高通量自动化组装的详细方案。我们已在一个由Biomek 3000液体处理机器人、台式离心机和板式热循环仪组成的最小硬件平台上实施了这些方案，并得到了一个名为AssemblyManager的软件工具的额外支持。这种方法能够以96%以上的成功率并行组装数百个无错误的大型DNA。
