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Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

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Directed Evolution

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2461))

Abstract

Protein engineering through directed evolutison is facilitated by the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries error-free and with minimal hands-on time, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

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References

  1. Packer M, Liu D (2015) Methods for the directed evolution of proteins. Nat Rev Genet 16:379–394. https://doi.org/10.1038/nrg3927

    Article  CAS  PubMed  Google Scholar 

  2. Cozens C, Pinheiro V (2018) XNA synthesis and reverse transcription by engineered thermophilic polymerases. Curr Protoc Chem Biol 10(3):e47. https://doi.org/10.1002/cpch.47

    Article  CAS  PubMed  Google Scholar 

  3. Zeymer C, Hilvert D (2018) Directed evolution of protein catalysts. Annu Rev Biochem 87:131–157. https://doi.org/10.1146/annurev-biochem-062917-012034

    Article  CAS  PubMed  Google Scholar 

  4. Lee J, Jeong E, Lee J et al (2018) Directed evolution of CRISPR-Cas9 to increase its specificity. Nat Commun 9(1):3048. https://doi.org/10.1038/s41467-018-05477-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Piatkevich K, Jung E, Straub C et al (2018) Publisher correction: a robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat Chem Biol 14(9):901. https://doi.org/10.1038/s41589-018-0023-6

    Article  CAS  PubMed  Google Scholar 

  6. Currin A, Swainston N, Day P, Kell D (2015) Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem Soc Rev 44(5):1172–1239. https://doi.org/10.1039/c4cs00351a

    Article  CAS  PubMed  Google Scholar 

  7. Cozens C, Pinheiro V (2018) Darwin assembly: fast, efficient, multi-site bespoke mutagenesis. Nucleic Acids Res 46(8):e51. https://doi.org/10.1093/nar/gky067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Siloto R, Weselake R (2012) Site saturation mutagenesis: methods and applications in protein engineering. Biocatal Agric Biotechnol 1(3):181–189. https://doi.org/10.1016/j.bcab.2012.03.010

    Article  CAS  Google Scholar 

  9. Sadowski MI, Grant C, Fell TS (2016) Harnessing QbD, programming languages, and automation for reproducible biology. Trends Biotechnol 34(3):214–227. https://doi.org/10.1016/j.tibtech.2015.11.006

    Article  CAS  PubMed  Google Scholar 

  10. Green M, Sambrook J (2016) Preparation of plasmid DNA by alkaline lysis with sodium dodecyl sulfate: Minipreps. Cold Spring Harb Protoc 2016(10). https://doi.org/10.1101/pdb.prot093344

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Acknowledgments

VBP thanks BBSRC (grant BB/N01023X/1—invivoXNA), ERC (grant 336936—HNAepisome) and the Rega Institute (grant ZL57031000). VBP and PH thank FWO (grant G0H7618N Odysseus).

Conflict of Interest

MK is an employee of Synthace, a for-profit company developing Antha.

Author information

Author notes

  1. S. Arangundy-Franklin

    Present address: Sangamo Therapeutics Inc., Brisbane, CA, USA

Authors and Affiliations

  1. Rega Institute, KU Leuven, Leuven, Belgium

    P. Handal-Marquez, D. Kestemont & V. B. Pinheiro

  2. Synthace Ltd., London, UK

    M. Koch

  3. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    S. Arangundy-Franklin

  4. Institute of Structural and Molecular Biology, University College London, London, UK

    V. B. Pinheiro

Authors
  1. P. Handal-Marquez
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  2. M. Koch
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  3. D. Kestemont
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  4. S. Arangundy-Franklin
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  5. V. B. Pinheiro
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Corresponding author

Correspondence to V. B. Pinheiro .

Editor information

Editors and Affiliations

  1. Manchester Institute of Biotechnology, University of Manchester, Manchester, UK

    Andrew Currin

  2. Manchester Institute of Biotechnology, University of Manchester, Manchester, UK

    Neil Swainston

1 Electronic Supplementary Material

Table S1

Oligonucleotides used for Darwin Assembly and Barcoding Libraries (DOCX 21 kb)

Table S2

Plasmid Sequences (DOCX 19 kb)

Supplementary File 1

Antha workflow and design file, Darwin oligo design, barcoding reconstruction script, and Kingfisher protocol (ZIP 45 kb)

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Handal-Marquez, P., Koch, M., Kestemont, D., Arangundy-Franklin, S., Pinheiro, V.B. (2022). Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries. In: Currin, A., Swainston, N. (eds) Directed Evolution. Methods in Molecular Biology, vol 2461. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2152-3_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2152-3_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2151-6

  • Online ISBN: 978-1-0716-2152-3

  • eBook Packages: Springer Protocols

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