Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

Colloms, S.D. , Merrick, C.A., Olorunniji, F.J., Stark, W.M. , Smith, M.C.M., Osbourn, A., Keasling, J.D. and Rosser, S.J. (2014) Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Research, 42(4), e23. (doi:10.1093/nar/gkt1101) (PMID:24225316) (PMCID:PMC3936721)

[img]
Preview
Text
87690.pdf - Published Version
Available under License Creative Commons Attribution.

1MB

Abstract

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Rosser, Professor Susan and Stark, Professor William and Olorunniji, Dr Femi and Colloms, Dr Sean
Authors: Colloms, S.D., Merrick, C.A., Olorunniji, F.J., Stark, W.M., Smith, M.C.M., Osbourn, A., Keasling, J.D., and Rosser, S.J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN:0305-1048
ISSN (Online):1362-4962
Copyright Holders:Copyright © 2013 The Authors
First Published:First published in Nucleic Acids Research 42(4):e23
Publisher Policy:Reproduced under a Creative Commons License

University Staff: Request a correction | Enlighten Editors: Update this record

Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
519111Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensemblesSusan RosserEngineering & Physical Sciences Research Council (EPSRC)EP/H019154/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
519113Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensemblesSusan RosserEngineering & Physical Sciences Research Council (EPSRC)EP/H019154/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
589141A platform for rapid and precise DNA module rearrangements in Synthetic BiologyWilliam StarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/K003356/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
589142A platform for rapid and precise DNA module rearrangements in Synthetic BiologyWilliam StarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/K003356/1RI MOLECULAR CELL & SYSTEMS BIOLOGY