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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Packer M, Liu D (2015) Methods for the directed evolution of proteins. Nat Rev Genet 16:379–394. https://doi.org/10.1038/nrg3927
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
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
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
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
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
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
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
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
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
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
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
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)
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
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