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

• Published in: Nucleic acids research Vol. 42; no. 4; p. e23
• Main Authors: Colloms, Sean D, Merrick, Christine A, Olorunniji, Femi J, Stark, W Marshall, Smith, Margaret C M, Osbourn, Anne, Keasling, Jay D, Rosser, Susan J
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.02.2014
• Subjects: 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
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkt1101

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.