| Autor: |
Potapov V; Research Department , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Ong JL; Research Department , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Kucera RB; Applications and Product Development , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Langhorst BW; Applications and Product Development , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Bilotti K; Research Department , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Pryor JM; Research Department , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Cantor EJ; Applications and Product Development , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Canton B; Ginkgo Bioworks , Boston , Massachusetts 02210 , United States., Knight TF; Ginkgo Bioworks , Boston , Massachusetts 02210 , United States., Evans TC Jr; Research Department , New England Biolabs , Ipswich , Massachusetts 01938 , United States., Lohman GJS; Research Department , New England Biolabs , Ipswich , Massachusetts 01938 , United States. |
| Abstrakt: |
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. |
