title: SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution.
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.
authors.affiliation: 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.
authors.forename: Andrew
authors.initials: A
authors.lastname: Currin
authors.affiliation: Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, UK School of Computer Science, The University of Manchester, Manchester M13 9PL, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Swainston
authors.affiliation: 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.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Day
authors.affiliation: 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.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Kell
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D019020:Directed Molecular Evolution / Q000295:instrumentation / Q000379:methods; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000138:chemical synthesis* / Q000235:genetics; D008995:Monoamine Oxidase / Q000138:chemical synthesis* / Q000235:genetics; D009841:Oligonucleotides / Q000138:chemical synthesis*; D019151:Peptide Library; D016133:Polymerase Chain Reaction
chemical_list: D009841:Oligonucleotides; D019151:Peptide Library; D049452:Green Fluorescent Proteins; D008995:Monoamine Oxidase
keywords: directed evolution; error correction; gene synthesis; protein libraries
references.citation: Chembiochem. 2011 Jul 4;12(10):1595-601
references.pmid: 21671328
references.citation: Nat Biotechnol. 2009 Dec;27(12):1151-62
references.pmid: 20010598
references.citation: Science. 2008 Feb 29;319(5867):1215-20
references.pmid: 18218864
references.citation: J Mol Biol. 2005 Jul 22;350(4):806-16
references.pmid: 15939434
references.citation: J Mol Biol. 1999 Jan 15;285(2):775-83
references.pmid: 9878443
references.citation: J Biochem Biophys Methods. 2008 Apr 24;70(6):820-2
references.pmid: 18272229
references.citation: Nucleic Acids Res. 2003 Nov 15;31(22):e143
references.pmid: 14602936
references.citation: Methods Enzymol. 2011;498:247-75
references.pmid: 21601681
references.citation: Nucleic Acids Res. 2010 May;38(8):2522-40
references.pmid: 20308161
references.citation: Org Biomol Chem. 2003 Dec 7;1(23):4133-7
references.pmid: 14685314
references.citation: Methods Mol Biol. 2012;852:11-21
references.pmid: 22328422
references.citation: Bioessays. 2012 Mar;34(3):236-44
references.pmid: 22252984
references.citation: Science. 2010 Jul 2;329(5987):52-6
references.pmid: 20488990
references.citation: Genome Res. 2004 Oct;14(10B):2020-8
references.pmid: 15489321
references.citation: Nucleic Acids Res. 2004 Jul 07;32(12):e98
references.pmid: 15240836
references.citation: Angew Chem Int Ed Engl. 2006 Feb 13;45(8):1236-41
references.pmid: 16411254
references.citation: Biotechnol Adv. 2011 Jan-Feb;29(1):156-63
references.pmid: 21034806
references.citation: J Theor Biol. 2009 Mar 7;257(1):131-41
references.pmid: 19073195
references.citation: Anal Biochem. 2004 Aug 15;331(2):401-3
references.pmid: 15265749
references.citation: Nucleic Acids Res. 2012 Feb;40(3):e23
references.pmid: 22127863
references.citation: J Theor Biol. 2005 Jun 21;234(4):497-509
references.pmid: 15808871
references.citation: FEMS Microbiol Rev. 2008 May;32(3):522-40
references.pmid: 18393987
references.citation: Nucleic Acids Res. 2002 May 15;30(10):e43
references.pmid: 12000848
references.citation: Nucleic Acids Res. 2005 Mar 30;33(6):e58
references.pmid: 15800209
references.citation: Nature. 1994 Aug 4;370(6488):389-91
references.pmid: 8047147
references.citation: Nucleic Acids Res. 2010 May;38(8):2663-75
references.pmid: 20385577
references.citation: Comput Struct Biotechnol J. 2012 Oct 22;2:e201209010
references.pmid: 24688651
references.citation: BMC Res Notes. 2010 Nov 09;3:291
references.pmid: 21062485
references.citation: Nat Chem Biol. 2009 Aug;5(8):567-73
references.pmid: 19620998
references.citation: Integr Biol (Camb). 2011 Feb;3(2):109-18
references.pmid: 21246151
references.citation: Nucleic Acids Res. 2014 Jul;42(Web Server issue):W395-400
references.pmid: 24782527
references.citation: Trends Biotechnol. 2008 Mar;26(3):132-8
references.pmid: 18222559
references.citation: Gene. 1995 Oct 16;164(1):49-53
references.pmid: 7590320
references.citation: Curr Biol. 1996 Feb 1;6(2):183-8
references.pmid: 8673465
references.citation: Biotechnol Adv. 2012 May-Jun;30(3):541-9
references.pmid: 21963685
references.citation: J Mol Biol. 2008 Dec 31;384(5):1218-31
references.pmid: 18951902
references.citation: Biotechnol Lett. 2012 Apr;34(4):721-8
references.pmid: 22187080
references.citation: Angew Chem Int Ed Engl. 2002 Sep 2;41(17):3177-80
references.pmid: 12207381
references.citation: Curr Opin Chem Biol. 2012 Aug;16(3-4):260-7
references.pmid: 22633067
references.citation: Angew Chem Int Ed Engl. 2006 Nov 27;45(46):7745-51
references.pmid: 17075931
references.citation: Nature. 2013 Feb 7;494(7435):77-80
references.pmid: 23354052
references.citation: Trends Biotechnol. 2012 Mar;30(3):147-54
references.pmid: 22209624
references.citation: Angew Chem Int Ed Engl. 2011 Jan 3;50(1):138-74
references.pmid: 20715024
references.citation: Nat Methods. 2008 Jan;5(1):37-9
references.pmid: 18037891
references.citation: Mol Biosyst. 2009 Jul;5(7):714-22
references.pmid: 19562110
references.citation: J Am Chem Soc. 2013 Jul 24;135(29):10863-9
references.pmid: 23808566
references.citation: Chemistry. 2006 Aug 7;12(23):6031-8
references.pmid: 16789057
references.citation: Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15440-5
references.pmid: 14657399
references.citation: Nat Methods. 2009 May;6(5):343-5
references.pmid: 19363495
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 15;110(3):E193-201
references.pmid: 23277561
references.citation: J Biomol Tech. 2007 Jul;18(3):147-9
references.pmid: 17595310
references.citation: Nat Biotechnol. 2010 Dec;28(12):1291-4
references.pmid: 21113166
references.citation: Eur J Biochem. 1985 Jul 1;150(1):1-5
references.pmid: 2410266
references.citation: Nat Protoc. 2007;2(4):891-903
references.pmid: 17446890