Nielsen Morten T, Madsen Karina M, Seppälä Susanna, Christensen Ulla, Riisberg Lone, Harrison Scott J, Møller Birger Lindberg, Nørholm Morten H H
†Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
ACS Synth Biol. 2015 Mar 20;4(3):274-82. doi: 10.1021/sb500055u. Epub 2014 Jun 24.
Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering.
分子克隆的标准化极大地促进了先进的DNA工程、部件共享以及诸如国际基因工程机器大赛（iGEM）这样的合作项目。所有这些特性都有助于利用基因组和RNA测序所提供的丰富遗传信息。标准化的代价是灵活性降低。我们通过制定一套设计原则来解决这一矛盾，旨在在保持克隆技术选择的高灵活性并将标准序列的影响降至最低的同时，最大限度地实现标准化。这些设计原则被应用于制定分子克隆流程，并迭代组装和优化用于在大肠杆菌中合成原卟啉IX的六基因途径。通过两个简单的工程和筛选循环就实现了当前的生产水平。这里定义的原则普遍适用，并简化了旨在构建或改造生物合成途径的项目的实验设计。
