Dataset Information

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 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.

SUBMITTER: Colloms SD

PROVIDER: S-EPMC3936721 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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

Colloms Sean D SD Merrick Christine A CA Olorunniji Femi J FJ Stark W Marshall WM Smith Margaret C M MC Osbourn Anne A Keasling Jay D JD Rosser Susan J SJ

Nucleic acids research 20131112 4

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 assembl ...[more]

PMID: 24225316

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Project description:SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (Int(SSV2)). Int(SSV2) alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1 to 80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165 to 328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81 to 164). It is worth noting that an N-terminally truncated mutant of Int(SSV2) (residues 81 to 328), which corresponded to the putative product of the 3'-end sequence of the Int(SSV2) gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length Int(SSV2) except for the slightly reduced recombination activity. Our results suggest that, unlike ? integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion.Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome. Many known archaeal viral integrases belong to a unique type, or the SSV type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision and reveal how different parts of the target DNA and the enzyme serve their roles in these processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase.

Dataset Information

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

Publications

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

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