Figure 2

The 2ab reaction. These are carried out in-vitro and used to make junctions between parts located on different assembly vectors. The choice of vector pair for each junction is determined by an assembly tree generated by AssemblyManager. AssemblyManager takes into account antibiotic resistance markers on both lefty and righty input plasmids and generates a binary tree containing legal lefty and righty pairs. In practice, lefty and righty elements are made by harvesting plasmids from E. coli strains that specifically methylate either BglII (part J72015) or BamHI (part J72015) restriction sites (methylated sites are shown in red and grayed over to indicate protection from digestion) [7, 8]. Isolated plasmids are then combined in one pot and digested with an enzyme cocktail containing BglII, BamHI and XhoI. Given the methylation state of each plasmid, lefties are cut with BamHI and XhoI, while righties are cut with BglII and XhoI. Following digestion, a ligation reaction recombines all fragments to generate a new composite part with a distinct permutation of antibiotic markers. The desired child product can then be selected away from undesired products, including parent plasmids, by growing it in the appropriate combination of antibiotics. Each new composite part can now be used iteratively in additional 2ab reactions to create progressively more complex parts.