PT  - JOURNAL ARTICLE
AU  - Marquez, P. Handal
AU  - Koch, M.
AU  - Kestemont, D.
AU  - Arangundy-Franklin, S.
AU  - Pinheiro, V. B.
TI  - Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries
AID  - 10.1101/2019.12.17.879486
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 2019.12.17.879486
4099  - http://biorxiv.org/content/early/2019/12/18/2019.12.17.879486.short
4100  - http://biorxiv.org/content/early/2019/12/18/2019.12.17.879486.full
AB  - Protein engineering through directed evolution facilitates 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 with minimal hands-on time and error-free, 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.