Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design

Large DNA constructs
(>10 kb) are invaluable tools for genetic
engineering and the development of therapeutics. However, the manufacture
of these constructs is laborious, often involving multiple hierarchical
rounds of preparation. To address this problem, we sought to test
whether Golden Gate assembly (GGA), an in vitro DNA
assembly methodology, can be utilized to construct a large DNA target
from many tractable pieces in a single reaction. While GGA is routinely
used to generate constructs from 5 to 10 DNA parts in one step, we
found that optimization permitted the assembly of >50 DNA fragments
in a single round. We applied these insights to genome construction,
successfully assembling the 40 kb T7 bacteriophage genome from up
to 52 parts and recovering infectious phage particles after cellular
transformation. The assembly protocols and design principles described
here can be applied to rapidly engineer a wide variety of large and
complex assembly targets.