SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution
SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution
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DOI：
10.1093/protein/gzu029
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发表时间：
2014-09-01
影响因子：
2.4
通讯作者：
Kell, Douglas B.
中科院分区：
文献类型：
--
作者：
Currin, Andrew;Swainston, Neil;Kell, Douglas B.
基因的从头合成在合成生物学研究中变得越来越普遍。然而，固有的错误率（由寡核苷酸合成期间发生的错误引入）将其在合成蛋白质文库中的使用限制为仅短基因。在这里，我们介绍SpeedyGenes，一种基于PCR的方法，用于合成不同的蛋白质文库，包括纠错程序，使大基因的有效合成直接用于功能筛选。首先，我们证明了一个准确的基因合成方法，通过合成和直接筛选（没有预先选择）一个747 bp的基因的绿色荧光蛋白（产生85%的荧光菌落）和一个更大的1518 bp的基因（单胺氧化酶，产生76%的菌落具有充分的催化活性，比以前的方法提高了4倍）。其次，我们表明SpeedyGenes可以容纳多个和组合的变体序列，同时保持有效的酶纠错，这对较大的基因尤其重要。在其第一次应用定向进化，我们证明了使用SpeedyGenes的MAO-N变体的大型文库的合成和筛选。使用该方法，在3天内合成、转化和筛选文库。重要的是，由于我们引入的每个突变都由寡核苷酸序列控制，SpeedyGenes能够合成大的，多样的，但受控的变体序列，用于定向进化。
The de novo synthesis of genes is becoming increasingly common in synthetic biology studies. However, the inherent error rate (introduced by errors incurred during oligonucleotide synthesis) limits its use in synthesising protein libraries to only short genes. Here we introduce SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enabling the efficient synthesis of large genes for use directly in functional screening. First, we demonstrate an accurate gene synthesis method by synthesising and directly screening (without pre-selection) a 747 bp gene for green fluorescent protein (yielding 85% fluorescent colonies) and a larger 1518 bp gene (a monoamine oxidase, producing 76% colonies with full catalytic activity, a 4-fold improvement over previous methods). Secondly, we show that SpeedyGenes can accommodate multiple and combinatorial variant sequences while maintaining efficient enzymatic error correction, which is particularly crucial for larger genes. In its first application for directed evolution, we demonstrate the use of SpeedyGenes in the synthesis and screening of large libraries of MAO-N variants. Using this method, libraries are synthesised, transformed and screened within 3 days. Importantly, as each mutation we introduce is controlled by the oligonucleotide sequence, SpeedyGenes enables the synthesis of large, diverse, yet controlled variant sequences for the purposes of directed evolution.