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.
Highlights
Synthetic biology seeks to engineer new function in biological systems for a variety of different applications
Bacteriophage fC31 integrase inserts the phage genome into its host’s DNA by recombining specific $46 bp attP and attB DNA sequences found in the phage and host genomes, respectively [20,21]
We have shown here that fC31 integrase can be used to rapidly insert one, three, four or five genes or other DNA fragments in any chosen arrangement directly into a SIRA plasmid vector
Summary
Synthetic biology seeks to engineer new function in biological systems for a variety of different applications. A key limitation in synthetic biology is the time taken to assemble genes or other DNA parts into new devices, pathways and systems, and to alter these assemblies once made. The junction sequences left by these methods make it difficult to change individual parts of the assembly without starting the assembly process from the beginning again. Most of these methods cannot produce large libraries of variant assemblies from large numbers of DNA parts for use in combinatorial assembly strategies. We illustrate the use of SIRA to assemble functional metabolic pathways from individual gene cassettes. The assembly process can incorporate multiple variants at all gene positions, randomize gene order and vary expression levels, allowing rapid pathway optimization
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