期刊
ACS SYNTHETIC BIOLOGY
卷 7, 期 11, 页码 2665-2674出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00333
关键词
DNA ligases; DNA ligase fidelity; Golden Gate assembly; DNA assembly; single-molecule sequencing
资金
- NEB
- Ginkgo Bioworks
- New England Biolabs
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.
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