Published online 29 April 2014 Nucleic Acids Research, 2014, Vol. 42, Web Server issue W395–W400 doi: 10.1093/nar/gku336 GeneGenie: optimized oligomer design for directed evolution Neil Swainston1,2,*, Andrew Currin1,3, Philip J. Day1,4 and Douglas B. Kell1,3

1Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, UK, 2School of Computer Science, The University of Manchester, Manchester M13 9PL, UK, 3School of Chemistry, The University of Manchester, Manchester M13 9PL, UK and 4Faculty of Medical and Human Sciences, The University of Manchester, Manchester M13 9PT, UK

Received January 30, 2014; Revised April 2, 2014; Accepted April 9, 2014

ABSTRACT GeneDesign (4) and DNAWorks (5). Each of these tools has their advantages: Gene Designer, for example, provides GeneGenie, a new online tool available at http://www. a comprehensive application for designing larger synthetic gene-genie.org, is introduced to support the de- systems whilst GeneDesign has recently been updated to al- sign and self-assembly of synthetic genes and con- low for the construction of entire chromosomes (6). How- structs. GeneGenie allows for the design of oligonu- ever, none of these packages supports the generation of vari- cleotide cohorts encoding the gene sequence opti- ant libraries to enable directed evolution studies. mized for expression in any suitable host through Consequently, GeneGenie, a new online tool available at an intuitive, easy-to-use web interface. The tool en- http://www.gene-genie.org, is introduced to support the de- sures consistent oligomer overlapping melting tem- sign of variant libraries of synthetic genes and constructs. peratures, minimizes the likelihood of misannealing, GeneGenie shares many features of existing optimization optimizes codon usage for expression in a selected software, allowing for the design of oligonucleotides en- coding the gene sequence responsible for the desired pro- host, allows for specification of forward and reverse tein sequence and optimized for expression in any suitable cloning sequences (for downstream ligation) and host through an intuitive, easy-to-use web interface. The also provides support for mutagenesis or directed tool ensures consistent oligomer overlap melting tempera- evolution studies. Directed evolution studies are en- tures, minimizes the likelihood of misannealing and opti- abled through the construction of variant libraries via mizes codon usage for expression in a selected host. the optional specification of ‘variant codons’, con- Output oligomers can be assembled using polymerase taining mixtures of bases, at any position. For exam- chain reaction (PCR)-based methods (7) and are fully com- ple, specifying the variant codon TNT (where N is any patible with our own optimized gene synthesis protocol de- nucleotide) will generate an equimolar mixture of the veloped alongside GeneGenie (A. Currin et al., manuscript codons TAT, TCT, TGT and TTT at that position, en- in preparation). These methods provide highly efficient as- coding a mixture of the amino acids Tyr, Ser, Cys and sembly, permitting expression and functional analysis of genes up to 2 kb in length before sequence verification. This Phe. This facility is demonstrated through the use of represents a significant improvement over currently estab- GeneGenie to develop and synthesize a library of en- lished direct gene synthesis methods. Using this integrated hanced green fluorescent protein variants. wet- and dry-lab approach, the successful synthesis and di- rect assay of enhanced green fluorescent protein (EGFP)8 ( ) is demonstrated. INTRODUCTION Novelties of GeneGenie include the specification of for- The de novo synthesis of genes is becoming increasingly es- ward and reverse cloning sequences, facilitating the ligation tablished in synthetic biology and biotechnology as a means of the designed gene into a vector and its subsequent ex- of controlling the specific assembly of amino acids produc- pression, and the op