Abstract
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.
Highlights
The ability to synthesise de novo and to assemble DNA molecules of any desired sequence is a fundamental feature of synthetic biology and biotechnology (Tian et al, 2009; Notka et al, 2011; Ma et al, 2012a)
We provide the first example of an integrated gene synthesis method that is fully complemented by an in silico design tool, GeneGenie (Swainston et al, 2014)
We describe an efficient method of gene synthesis that requires fewer steps than those described previously, thereby providing the opportunity to assemble large and accurate constructs
Summary
The ability to synthesise de novo and to assemble DNA molecules of any desired sequence is a fundamental feature of synthetic biology and biotechnology (Tian et al, 2009; Notka et al, 2011; Ma et al, 2012a). Each copy of the synthesised gene often encodes randomly placed mutations or (more commonly) base insertions or deletions, with a greater number incorporated as the length of the synthesised nucleic acid increases These errors require removal to realise the desired DNA sequence (Xiong et al, 2008; Ma et al, 2012b), a process that creates a significant bottleneck in efficient gene synthesis. Various strategies have been employed to reduce encoded errors, including use of mismatch-binding proteins (Carr and Church, 2009), enzymatic mismatch cleavage (Fuhrmann et al, 2005; Saaem et al, 2012), site-directed mutagenesis (Xiong et al, 2008), synthesis of higher quality oligonucleotides (LeProust et al, 2010) and retrieval of sequence-verified molecules using pyrosequencing (Matzas et al, 2010) None of these methods is entirely satisfactory, nor (in particular) is applicable to the production of genetic libraries
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