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.

Bricks and blueprints: methods and standards for DNA assembly

“…By synthetically generating four additional orthogonal att recombination sequences, Gateway has also recently been expanded to enable the cloning of multiple parts simultaneously 41 . Similar non-commercial systems have also been developed that use alternative phage integrases, including phiBT1 and phiC31 42,43 . For all these methods, reactions at recombined att sites can be reversed by additionally providing either an excisionase (a bacteriophage excision protein) or a recombination directionality factor (RDF), which is an accessory protein that in combination with the integrase reverses the reaction and leads to excision rather than integration.…”

“…For all these methods, reactions at recombined att sites can be reversed by additionally providing either an excisionase (a bacteriophage excision protein) or a recombination directionality factor (RDF), which is an accessory protein that in combination with the integrase reverses the reaction and leads to excision rather than integration. For phiC31 integrase-based assembly, excision allows quick replacement of a single part within an already assembled construct, enabling the insertion of an alternative part or of a multi-part fragment that further expands the construct 43 .…”

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

“…Serine integrases are being used in a variety of genetic engineering and synthetic biology applications (15)(16)(17)(18)(19)(20)(21)(22). A major attribute of serine integrases is that they perform unidirectional recombination, as regulated by their dedicated RDF.…”

Mechanistic modelling of a recombinase‐based two‐input temporal logic gate

“…Tyrosine recombinase systems that are not dependent on host cofactors are bidirectional, and are, therefore, incapable of highly efficient switching since there can be no guarantee that an induced transition to a desired DNA state would be effectively maintained without unwanted transitioning back to the original state [15]. In contrast, serine recombinases do not require such cofactors and have been used effectively to perform highly efficient unidirectional gene assembly and modification [16]. This has led to the construction of a rewritable recombinase addressable data (RAD) module exhibiting passive information storage within a chromosome [9].…”