University College London, Gower Street, London WC1E 6BT, UK.
Institute of Structural and Molecular Biology, Birkbeck College, University of London, Malet Street WC1E 7HX, UK.
Nucleic Acids Res. 2018 May 4;46(8):e51. doi: 10.1093/nar/gky067.
Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations - particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.
为了实现特定功能和生物技术应用而对蛋白质进行工程设计，几乎都需要（或至少受益于）对非连续残基进行多次突变。存在几种用于多位点定向诱变的方法，但仍需要快速、简单的方法来有效地引入此类突变，尤其是要生成用于定向进化的大型高质量文库。在这里，我们介绍 Darwin Assembly，它可以提供高质量的文库，转化效率超过 108 个转化体，靶向多个（>10）远端位点，野生型污染最小（<总群体的 0.25%），从纯化质粒到文库转化只需一个工作日。我们通过氯霉素乙酰转移酶的全基因密码子重排证明了其功效，在 KOD DNA 聚合酶中一次反应突变 19 个密码子，并在 T7 RNA 聚合酶和 Tgo DNA 聚合酶中生成高质量的多位点文库。Darwin Assembly 使用市售酶，易于自动化，为高度复杂和可定制的文库生成提供了具有成本效益的途径。
