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High fidelity one-pot DNA assembly using orthogonal serine integrases

Abioye, J, Lawson-Williams, M, Lecanda, A, Calhoon, B, McQue, AL, Colloms, SD, Stark, WM and Olorunniji, FJ (2022) High fidelity one-pot DNA assembly using orthogonal serine integrases. Biotechnology Journal. ISSN 1860-6768

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Open Access URL: https://doi.org/10.1002/biot.202200411 (Published version)


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: Article
Uncontrolled Keywords: SIRA; genome editing; large serine integrases (LSIs); site-specific recombination; synthetic biology; 1002 Environmental Biotechnology; 1003 Industrial Biotechnology; 1004 Medical Biotechnology; Biotechnology
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
R Medicine > RM Therapeutics. Pharmacology
Divisions: Pharmacy & Biomolecular Sciences
Publisher: Wiley
SWORD Depositor: A Symplectic
Date Deposited: 01 Mar 2023 11:56
Last Modified: 01 Mar 2023 12:00
DOI or ID number: 10.1002/biot.202200411
URI: https://researchonline.ljmu.ac.uk/id/eprint/19000
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