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)
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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 Marshall 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 > School of Molecular Biosciences |
| 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 |
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