Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination
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 C31 integrase. Using six orthogonal attP/attB recombination site pair...
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| Published in | Nucleic acids research Vol. 42; no. 4; p. e23 |
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| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Oxford University Press
01.02.2014
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| Subjects | |
| Online Access | Get full text |
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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 C31 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. C31 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. |
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| AbstractList | 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 C31 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. C31 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. 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. 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. 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. |
| Author | Keasling, Jay D Colloms, Sean D Olorunniji, Femi J Stark, W Marshall Smith, Margaret C M Osbourn, Anne Merrick, Christine A Rosser, Susan J |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/24225316$$D View this record in MEDLINE/PubMed https://www.osti.gov/servlets/purl/1625515$$D View this record in Osti.gov |
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| Snippet | Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid... |
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| SubjectTerms | Bacteriophages - enzymology BASIC BIOLOGICAL SCIENCES Biochemistry & Molecular Biology Biosynthetic Pathways - genetics Cloning, Molecular - methods Gene Order Integrases - metabolism Metabolic Engineering - methods Metabolic Networks and Pathways - genetics Methods Online Recombination, Genetic Ribosomes - metabolism Synthetic Biology - methods Synthetic Biology and Assembly Cloning |
| Title | Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination |
| URI | https://www.ncbi.nlm.nih.gov/pubmed/24225316 https://search.proquest.com/docview/1503552062 https://www.osti.gov/servlets/purl/1625515 https://pubmed.ncbi.nlm.nih.gov/PMC3936721 |
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