Imperial College Centre for Synthetic Biology, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.
Nucleic Acids Res. 2019 Feb 20;47(3):e17. doi: 10.1093/nar/gky1182.
DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.
DNA 组装允许快速可靠地组装单个 DNA 构建体或文库。大多数方法要么：(i) 模块化，易于扩展，适用于组合组装，但会留下不理想的“疤痕”序列；要么 (ii) 定制（非模块化），无疤痕，但不太适合构建组合文库。两者在代谢工程方面都有局限性。为了克服这种权衡，我们设计了 Start-Stop 组装，这是一种多部分、模块化的 DNA 组装方法，既无功能疤痕，又适用于组合组装。至关重要的是，使用与起始和终止密码子相对应的 3 bp 突出端将编码序列组装成表达单元，从而避免在敏感的编码序列边界处产生疤痕。在此概念的基础上，设计并实现了一个完整的 DNA 组装框架，允许从多达 60 个部分（或混合物）中组装多达 15 个基因；单顺反子、操纵子或混合配置；以及新的简化组装层次结构，最大限度地减少向量的数量。每个生物体只需要一个目的载体，这反映了我们对不同生物体代谢工程系统的优化。通过在大肠杆菌中组合组装类胡萝卜素途径来展示 Start-Stop 组装的代谢工程，产生了广泛的类胡萝卜素生产和菌落大小表型，表明对设计空间的预期探索。
