Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark.
ACS Synth Biol. 2024 Mar 15;13(3):963-968. doi: 10.1021/acssynbio.3c00665. Epub 2024 Mar 4.
Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.
近年来，基因合成的效率有了很大的提高，但在重复序列方面仍受到限制，这会导致 DNA 合成供应商合成失败或延迟。这对于合成生物学的发展来说是一个主要障碍，因为重复元件越来越多地被用于基因回路的设计和生物分子纳米结构的设计。在这里，我们描述了一种用于组装具有重复元件的小合成基因的方法：首先，将感兴趣的基因分割成小的合成子，每个合成子长 80 个碱基对，两侧为 Golden-Gate 兼容的突出端。然后，通过寡核苷酸延伸来制作合成子，最后通过 Golden Gate 组装将它们组装成一个合成基因。我们通过构建具有重复元件的八个具有挑战性的基因来证明该方法的有效性，例如，多个 RNA 适体和具有多个相同适体的 RNA 折纸支架的重复。这些基因的大小从 133 到 456 个碱基对不等，组装的保真度高达 87.5%。该方法是为了方便我们自己的特定研究而开发的，但可能对构建具有挑战性和重复的基因具有普遍适用性，因此是分子克隆工具箱的一个有价值的补充。
