Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly

Synthetic
biology relies on the manufacture of large and complex
DNA constructs from libraries of genetic parts. Golden Gate and other
Type IIS restriction enzyme-dependent DNA assembly methods enable
rapid construction of genes and operons through one-pot, multifragment
assembly, with the ordering of parts determined by the ligation of
Watson–Crick base-paired overhangs. However, ligation of mismatched
overhangs leads to erroneous assembly, and low-efficiency Watson Crick
pairings can lead to truncated assemblies. Using sets of empirically
vetted, high-accuracy junction pairs avoids this issue but limits
the number of parts that can be joined in a single reaction. Here,
we report the use of comprehensive end-joining ligation fidelity and
bias data to predict high accuracy junction sets for Golden Gate assembly.
The ligation profile accurately predicted junction fidelity in ten-fragment
Golden Gate assembly reactions and enabled accurate and efficient
assembly of a lac cassette from up to 24-fragments
in a single reaction.