Golden Gate assembly of BioBrick-compliant parts using Type II restriction endonucleases

Method summary Plasmids that comply with the BioBrick standard are used to transform Escherichia coli cells carrying DNA methyltransferase expression vectors. The methylated plasmids are purified and digested with Type II restriction endonucleases. All restriction fragments are ligated to each other without purification, but unwanted ligation products are linearized in a second round of restriction digestion before the mixture is used to transform E. coli. Aims: New methods of DNA recombination that capture the principal advantages of the BioBrick standard (ease of design) and Golden Gate assembly (decreased labor) are demonstrated here. Methods & materials: Both methods employ DNA methyltransferase expression vectors, available from Addgene, that protect selected sites on different plasmids from particular Type II restriction endonucleases. No other reagents are required. Results: The 4R/2M discontinuous DNA assembly is more efficient (produces more desired recombinant plasmids) and as specific (produces few undesired recombination products) as conventional subcloning. The 5RM continuous DNA assembly is approximately as efficient and specific as conventional Golden Gate assembly, even though in vivo methylation of one plasmid is incomplete. Conclusion: Both methylase-assisted methods streamline BioBrick assembly workflows without complicating the design of synthetic parts.