Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA and Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
Nucleic Acids Res. 2014 Jan;42(1):681-9. doi: 10.1093/nar/gkt860. Epub 2013 Sep 26.
In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.
体外重组方法使人们能够一步从多个部分构建大型 DNA 序列。尽管合成生物电路原则上可以以相同的方式组装，但它们通常包含重复的序列元件，如标准启动子和终止子，这些元件会干扰同源重组。在这里，我们使用一种计算方法来设计合成的、无生物活性的独特核苷酸序列（UNSes），以促进准确的有序组装。重要的是，我们设计的 UNSes 使得可以组装带有重复终止子和绝缘子序列的部分，从而在细菌和哺乳动物细胞中创建绝缘功能基因回路。我们使用 UNS 引导的组装来构建具有重复启动子-基因-终止子部分的重复序列，系统地改变基因表达，以优化脱氧胆酸杆菌生物合成途径在大肠杆菌中的生产。然后，我们使用该系统构建了用于基因组整合到胚胎干细胞中的复杂真核 AND 逻辑门。构建是通过使用一系列标准化的带有 UNS 的 BioBrick 兼容载体来完成的，这些载体能够进行模块化组装并方便地重复使用各个部分。UNSes 引导的等温组装广泛适用于基因回路的构建和优化，特别是那些需要紧密绝缘的回路，如复杂的生物合成途径、传感器、计数器和逻辑门。
