Sequeira Ana Filipa, Brás Joana L A, Fernandes Vânia O, Guerreiro Catarina I P D, Vincentelli Renaud, Fontes Carlos M G A
Faculdade de Medicina Veterinária, Centro Interdisciplinar de Investigação em Sanidade Animal (CIISA), Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal.
NZYTech Genes & Enzymes, Estrada do Paço do Lumiar, Campus do Lumiar, Edifício E, r/c, 1649-038, Lisbon, Portugal.
Methods Mol Biol. 2017;1620:113-128. doi: 10.1007/978-1-4939-7060-5_7.
Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. Here, we describe a high-throughput platform to design and produce multiple synthetic genes (<500 bp) for recombinant expression in Escherichia coli. This pipeline includes an innovative codon optimization algorithm that designs DNA sequences to maximize heterologous protein production in different hosts. The platform is based on a simple gene synthesis method that uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides. This technology incorporates an accurate, automated and cost-effective ligase-independent cloning step to directly integrate the synthetic genes into an effective E. coli expression vector. High-throughput production of synthetic genes is of increasing relevance to allow exploring the biological function of the extensive genomic and meta-genomic information currently available from various sources.
基因合成正成为重组DNA技术诸多领域的一项重要工具，包括重组蛋白生产。从头合成基因正在迅速取代传统的克隆和诱变程序，并能够生成无可用模板的核酸。在此，我们描述了一个高通量平台，用于设计和生产多个用于在大肠杆菌中重组表达的合成基因（<500 bp）。该流程包括一种创新的密码子优化算法，该算法设计DNA序列以在不同宿主中最大化异源蛋白产量。该平台基于一种简单的基因合成方法，该方法使用基于PCR的方案从重叠寡核苷酸库中组装合成DNA。这项技术包含一个准确、自动化且经济高效的不依赖连接酶的克隆步骤，可将合成基因直接整合到有效的大肠杆菌表达载体中。合成基因的高通量生产对于探索目前可从各种来源获得的大量基因组和宏基因组信息的生物学功能越来越重要。
