Large-scale gene synthesis in tiny droplets Gene synthesis technology is important for functional characterization of DNA sequences and for the development of synthetic biology. However, current methods are limited by their low scalability and high cost. Plesa et al. developed a gene synthesis method, DropSynth, which uses barcoded beads to concentrate oligos and subsequently assemble them into synthetic genes within picoliter emulsion droplets. DropSynth allows generation of large libraries of thousands of genes and functional testing of all possible mutations of a particular sequence. Science, this issue p. 343 A gene synthesis method, DropSynth, allows for the synthesis and characterization of thousands of pooled genes. Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene’s assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in Escherichia coli. We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.

中文翻译：

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乳液中的多重基因合成用于探索蛋白质功能景观

微小液滴中的大规模基因合成基因合成技术对于DNA序列的功能表征和合成生物学的发展具有重要意义。然而，当前的方法因其可扩展性低和成本高而受到限制。普莱萨等人。开发了一种基因合成方法 DropSynth，该方法使用条形码珠来浓缩寡核苷酸，然后将它们组装成皮升乳液液滴内的合成基因。DropSynth 允许生成包含数千个基因的大型文库，并对特定序列的所有可能突变进行功能测试。科学，本期第 14 页。343 基因合成方法 DropSynth 可以合成和表征数千个汇集基因。提高我们构建和功能表征 DNA 序列的能力将广泛加速生物学的进步。在这里，我们介绍 DropSynth，这是一种可扩展、低成本的方法，可以以汇集（多重）方式构建数千个定义的基因长度构建体。DropSynth 使用条形码珠子库来提取基因组装所需的寡核苷酸，然后在油包水乳液中进行处理和组装。我们使用 DropSynth 成功构建了 7000 多个合成基因，这些基因编码大肠杆菌中两个必需基因的系统发育多样性同源物。我们测试了磷酸泛硫氨酸腺苷酸转移酶同系物与多重敲除大肠杆菌菌株互补的能力，揭示了核心功能基序和同系物不相容的潜在原因。DropSynth 与多重功能分析相结合使我们能够以前所未有的规模合理地探索序列功能关系。