Woodruff Lauren B A, Gorochowski Thomas E, Roehner Nicholas, Mikkelsen Tarjei S, Densmore Douglas, Gordon D Benjamin, Nicol Robert, Voigt Christopher A
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Nucleic Acids Res. 2017 Feb 17;45(3):1553-1565. doi: 10.1093/nar/gkw1226.
Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of ∼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithm-guided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated.
基因设计可以由数十个基因和数百个基因元件组成。在评估一个设计后，希望在不承担从头开始构建过程的成本和负担的情况下进行修改。在此，我们报告了一个两步过程，其中一个大的设计空间被划分为复合元件的深度文库，从中检索个体并进行组装以构建最终构建体。这些文库通过多重组装构建，并使用下一代测序进行测序。每个文库由约20兆碱基（Mb）的多达5000个独特且经过序列验证的复合元件组成，这些元件带有条形码以便通过聚合酶链反应（PCR）进行检索。这种方法应用于一个16个基因的固氮途径，该途径被分解为总共包含55848个复合元件（71.0 Mb）的文库。这些文库涵盖了一个巨大的设计空间（1043种可能的23千碱基（kb）构建体），从中构建了一个由算法引导的192个成员的4.5 Mb文库。接下来，基于10个阻遏物（或非/非门）的所有1030种可能的遗传电路在文库中进行编码，其中每个阻遏物与输入启动子的所有排列融合。这些证明了多重技术可以应用于涵盖整个设计空间，从中可以获取和评估个体。
