PMID- 36504358
OWN - NLM
STAT- MEDLINE
DCOM- 20230302
LR  - 20240306
IS  - 1860-7314 (Electronic)
IS  - 1860-6768 (Linking)
VI  - 18
IP  - 3
DP  - 2023 Mar
TI  - High fidelity one-pot DNA assembly using orthogonal serine integrases.
PG  - e2200411
LID - 10.1002/biot.202200411 [doi]
AB  - 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, varphiR4, 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 beta-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.
CI  - (c) 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
FAU - Abioye, Jumai
AU  - Abioye J
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
FAU - Lawson-Williams, Makeba
AU  - Lawson-Williams M
AD  - School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John 
      Moores University, Liverpool, UK.
FAU - Lecanda, Alicia
AU  - Lecanda A
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
FAU - Calhoon, Brecken
AU  - Calhoon B
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
FAU - McQue, Arlene L
AU  - McQue AL
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
FAU - Colloms, Sean D
AU  - Colloms SD
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
FAU - Stark, W Marshall
AU  - Stark WM
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
FAU - Olorunniji, Femi J
AU  - Olorunniji FJ
AUID- ORCID: 0000-0001-9389-2981
AD  - School of Molecular Biosciences, University of Glasgow, Glasgow, UK.
AD  - School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John 
      Moores University, Liverpool, UK.
LA  - eng
GR  - MC_PC_16077/MRC_/Medical Research Council/United Kingdom
PT  - Journal Article
DEP - 20221228
PL  - Germany
TA  - Biotechnol J
JT  - Biotechnology journal
JID - 101265833
RN  - EC 2.7.7.- (Integrases)
RN  - 452VLY9402 (Serine)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - *Integrases/genetics
MH  - Serine/metabolism
MH  - DNA/genetics
MH  - Plasmids/genetics
MH  - *Bacteriophages/genetics/metabolism
OTO - NOTNLM
OT  - SIRA
OT  - genome editing
OT  - large serine integrases (LSIs)
OT  - site-specific recombination
OT  - synthetic biology
EDAT- 2022/12/13 06:00
MHDA- 2023/03/03 06:00
CRDT- 2022/12/12 10:31
PHST- 2022/12/08 00:00 [revised]
PHST- 2022/08/18 00:00 [received]
PHST- 2022/12/09 00:00 [accepted]
PHST- 2022/12/13 06:00 [pubmed]
PHST- 2023/03/03 06:00 [medline]
PHST- 2022/12/12 10:31 [entrez]
AID - 10.1002/biot.202200411 [doi]
PST - ppublish
SO  - Biotechnol J. 2023 Mar;18(3):e2200411. doi: 10.1002/biot.202200411. Epub 2022 Dec 
      28.