High fidelity one‐pot DNA assembly using orthogonal serine integrases

Abioye, J., Lawson‐Williams, M., Lecanda, A., Calhoon, B., McQue, A. L., Colloms, S. D. , Stark, W. M. and Olorunniji, F. J. (2023) High fidelity one‐pot DNA assembly using orthogonal serine integrases. Biotechnology Journal, 18(3), 2200411. (doi: 10.1002/biot.202200411) (PMID:36504358)

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Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. Methods and Major Results: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up “poly-part” gene assembly and editing. Conclusions and Implications: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.

Item Type:Articles
Additional Information:This work was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/003356/1) and Medical Research Council Proximity (MC PC 16077).
Glasgow Author(s) Enlighten ID:Stark, Professor Marshall and Olorunniji, Dr Femi and Colloms, Dr Sean and McQue, Mrs Arlene and Abioye, Jumai
Authors: Abioye, J., Lawson‐Williams, M., Lecanda, A., Calhoon, B., McQue, A. L., Colloms, S. D., Stark, W. M., and Olorunniji, F. J.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Biotechnology Journal
ISSN (Online):1860-7314
Published Online:12 December 2022
Copyright Holders:Copyright © 2022 The Authors
First Published:First published in Biotechnology Journal 18(3): 2200411
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
167747A platform for rapid and precise DNA module rearrangements in Synthetic BiologyWilliam StarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/K003356/1MCSB - Molecular Genetics