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

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.

Authors:
 [1];  [1];  [1];  [1];  [2];  [3];  [4];  [1]
  1. Univ. of Glasgow, Scotland (United Kingdom)
  2. Univ. of York (United Kingdom)
  3. Norwich Research Park, Norwich (United Kingdom)
  4. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
OSTI Identifier:
1625515
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 42; Journal Issue: 4; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biochemistry & Molecular Biology; Synthetic Biology and Assembly Cloning

Citation Formats

Colloms, Sean D., Merrick, Christine A., Olorunniji, Femi J., Stark, W. Marshall, Smith, Margaret C. M., Osbourn, Anne, Keasling, Jay D., and Rosser, Susan J. Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. United States: N. p., 2013. Web. doi:10.1093/nar/gkt1101.
Colloms, Sean D., Merrick, Christine A., Olorunniji, Femi J., Stark, W. Marshall, Smith, Margaret C. M., Osbourn, Anne, Keasling, Jay D., & Rosser, Susan J. Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. United States. https://doi.org/10.1093/nar/gkt1101
Colloms, Sean D., Merrick, Christine A., Olorunniji, Femi J., Stark, W. Marshall, Smith, Margaret C. M., Osbourn, Anne, Keasling, Jay D., and Rosser, Susan J. Tue . "Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination". United States. https://doi.org/10.1093/nar/gkt1101. https://www.osti.gov/servlets/purl/1625515.
@article{osti_1625515,
title = {Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination},
author = {Colloms, Sean D. and Merrick, Christine A. and Olorunniji, Femi J. and Stark, W. Marshall and Smith, Margaret C. M. and Osbourn, Anne and Keasling, Jay D. and Rosser, Susan J.},
abstractNote = {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.},
doi = {10.1093/nar/gkt1101},
journal = {Nucleic Acids Research},
number = 4,
volume = 42,
place = {United States},
year = {Tue Nov 12 00:00:00 EST 2013},
month = {Tue Nov 12 00:00:00 EST 2013}
}

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Engineering the lycopene synthetic pathway in E. coli by comparison of the carotenoid genes of Pantoea agglomerans and Pantoea ananatis
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Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids
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DNA assembly for synthetic biology: from parts to pathways and beyond
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Gated rotation mechanism of site-specific recombination by  C31 integrase
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In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family
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Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids
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DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways
journal, December 2008

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Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides
journal, September 2009


SLiCE: a novel bacterial cell extract-based DNA cloning method
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DNA Cloning Using In Vitro Site-Specific Recombination
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A One Pot, One Step, Precision Cloning Method with High Throughput Capability
journal, November 2008


Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes
journal, May 2009


Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways
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