September 22, 2019  |  

Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly.

Authors: Potapov, Vladimir and Ong, Jennifer L and Kucera, Rebecca B and Langhorst, Bradley W and Bilotti, Katharina and Pryor, John M and Cantor, Eric J and Canton, Barry and Knight, Thomas F and Evans, Thomas C and Lohman, Gregory J S

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.

Journal: ACS synthetic biology
DOI: 10.1021/acssynbio.8b00333
Year: 2018

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