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

Colloms, Sean D.; Merrick, Christine A.; Olorunniji, Femi J.; Stark, W. Marshall; Smith, Margaret C.  M.; Osbourn, Anne; Keasling, Jay D.; Rosser, Susan J.

doi:10.1093/nar/gkt1101

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

Journal Article · Mon Nov 11 23:00:00 EST 2013 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkt1101· OSTI ID:1625515

Colloms, Sean D. ^[1]; Merrick, Christine A. ^[2]; Olorunniji, Femi J. ^[2]; Stark, W. Marshall ^[2]; Smith, Margaret C. M. ^[3]; Osbourn, Anne ^[4]; Keasling, Jay D. ^[5]; Rosser, Susan J. ^[2]

Univ. of Glasgow, Scotland (United Kingdom); DOE/OSTI
Univ. of Glasgow, Scotland (United Kingdom)
Univ. of York (United Kingdom)
Norwich Research Park, Norwich (United Kingdom)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1625515

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 4 Vol. 42; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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