Abstract
This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement, and domain insertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restriction endonuclease generates blunt-end random breaks at a frequency of one per target gene and the concomitant loss of 3 bp. Self-ligation or insertion of another DNA cassette results in the sampling of trinucleotide deletion or trinucleotide substitution/domain insertion, respectively.
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References
Cobb RE, Si T, Zhao H (2012) Directed evolution: an evolving and enabling synthetic biology tool. Curr Opin Chem Biol 16:285–291
Jackel C, Kast P, Hilvert D (2008) Protein design by directed evolution. Annu Rev Biophys 37:153–173
Lutz S, Patrick WM (2004) Novel methods for directed evolution of enzymes: quality, not quantity. Curr Opin Biotechnol 15:291–297
Neylon C (2004) Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res 32:1448–1459
Brustad EM, Arnold FH (2011) Optimizing non-natural protein function with directed evolution. Curr Opin Chem Biol 15:201–210
Koide S (2009) Generation of new protein functions by nonhomologous combinations and rearrangements of domains and modules. Curr Opin Biotechnol 20:398–404
Chothia C, Gough J, Vogel C, Teichmann SA (2003) Evolution of the protein repertoire. Science 300:1701–1703
Taylor MS, Ponting CP, Copley RR (2004) Occurrence and consequences of coding sequence insertions and deletions in Mammalian genomes. Genome Res 14:555–566
Shortle D, Sondek J (1995) The emerging role of insertions and deletions in protein engineering. Curr Opin Biotechnol 6:387–393
Jones DD (2005) Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 β-lactamase to an amino acid deletion. Nucleic Acids Res 33:e80
Simm AM, Baldwin AJ, Busse K, Jones DD (2007) Investigating protein structural plasticity by surveying the consequence of an amino acid deletion from TEM-1 β-lactamase. FEBS Lett 581:3904–3908
Baldwin AJ, Arpino JA, Edwards WR, Tippmann EM, Jones DD (2009) Expanded chemical diversity sampling through whole protein evolution. Mol Biosyst 5:764–766
Baldwin AJ, Busse K, Simm AM, Jones DD (2008) Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx). Nucleic Acids Res 36:e77
Arpino JA, Czapinska H, Piasecka A, Edwards WR, Barker P, Gajda MJ, Bochtler M, Jones DD (2012) Structural basis for efficient chromophore communication and energy transfer in a constructed didomain protein scaffold. J Am Chem Soc 134:13632–13640
Edwards WR, Busse K, Allemann RK, Jones DD (2008) Linking the functions of unrelated proteins using a novel directed evolution domain insertion method. Nucleic Acids Res 36:e78. doi:10.1093/nar/gkn363
Edwards WR, Williams AJ, Morris JL, Baldwin AJ, Allemann RK, Jones DD (2010) Regulation of β-lactamase activity by remote binding of haem: functional coupling of unrelated proteins through domain insertion. Biochemistry 49:6541–6549
Haapa S, Taira S, Heikkinen E, Savilahti H (1999) An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications. Nucleic Acids Res 27:2777–2784
Fastrez J (2009) Engineering allosteric regulation into biological catalysts. Chembiochem 10:2824–2835
Ferraz RM, Vera A, Aris A, Villaverde A (2006) Insertional protein engineering for analytical molecular sensing. Microb Cell Fact 5:15
Ostermeier M (2005) Engineering allosteric protein switches by domain insertion. Protein Eng Des Sel 18:359–364
Acknowledgments
The authors would like to thank the BBSRC (BB/H003746, BB/E001084, BB/FOF/263, BB/E007384), MRC DPFS (G0900868), Merck KGaA, Wellcome (084542/Z/07/Z), and Nuffield Foundation for supporting this work.
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Jones, D.D., Arpino, J.A.J., Baldwin, A.J., Edmundson, M.C. (2014). Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolution. In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_11
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DOI: https://doi.org/10.1007/978-1-4939-1053-3_11
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