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

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Abstract
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, multi-fragment 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.
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  • New England Biolabs
  • Ginkgo Bioworks

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