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Protein Eng Des Sel. 2014 Sep;27(9):273-80. doi: 10.1093/protein/gzu029. Epub 2014 Aug 9.

SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution.

Author information

1
Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, UK School of Chemistry, The University of Manchester, Manchester M13 9PL, UK andrew.currin@manchester.ac.uk dbk@manchester.ac.uk.
2
Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, UK School of Computer Science, The University of Manchester, Manchester M13 9PL, UK.
3
Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, UK Faculty of Medical and Human Sciences, The University of Manchester, Manchester M13 9PT, UK.

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.

KEYWORDS:

directed evolution; error correction; gene synthesis; protein libraries

PMID:
25108914
PMCID:
PMC4140418
DOI:
10.1093/protein/gzu029
[Indexed for MEDLINE]
Free PMC Article

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