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Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling

Potapov Vladimir, Jennifer L. Ong, Rebecca B. Kucera, Bradley W. Langhorst, Katharina Bilotti, John M. Pryor, Eric J. Cantor, Barry Canton, Thomas F. Knight, Thomas C. Evans Jr., Gregory J. S. Lohman
doi: https://doi.org/10.1101/322297
Potapov Vladimir
1Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Jennifer L. Ong
1Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Rebecca B. Kucera
2Applications and Product Development, New England Biolabs, Ipswich, MA, 01938, USA
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Bradley W. Langhorst
2Applications and Product Development, New England Biolabs, Ipswich, MA, 01938, USA
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Katharina Bilotti
1Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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John M. Pryor
1Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Eric J. Cantor
2Applications and Product Development, New England Biolabs, Ipswich, MA, 01938, USA
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Barry Canton
3Ginkgo Bioworks, Boston, MA, 02210, USA
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Thomas F. Knight
3Ginkgo Bioworks, Boston, MA, 02210, USA
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Thomas C. Evans Jr.
1Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Gregory J. S. Lohman
1Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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  • For correspondence: lohman@neb.com
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ABSTRACT

Modern synthetic biology depends on the manufacture of large DNA constructs from libraries of genes, regulatory elements or other genetic parts. Type IIS restriction enzyme-dependent DNA assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly, facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has typically been accomplished by using small sets of empirically vetted junction pairs, limiting the number of parts that can be joined in a single reaction. Here, we report the use of a comprehensive method for profiling end-joining ligation fidelity and bias to predict highly accurate sets of connections for ligation-based DNA assembly methods. This data set allows quantification of sequence-dependent ligation efficiency and identification of mismatch-prone pairings. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions, and enabled efficient assembly of a lac cassette from up to 24-fragments in a single reaction. Application of the ligation fidelity profile to inform choice of junctions thus enables highly flexible assembly design, with >20 fragments in a single reaction.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted May 15, 2018.
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Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling
Potapov Vladimir, Jennifer L. Ong, Rebecca B. Kucera, Bradley W. Langhorst, Katharina Bilotti, John M. Pryor, Eric J. Cantor, Barry Canton, Thomas F. Knight, Thomas C. Evans Jr., Gregory J. S. Lohman
bioRxiv 322297; doi: https://doi.org/10.1101/322297
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Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling
Potapov Vladimir, Jennifer L. Ong, Rebecca B. Kucera, Bradley W. Langhorst, Katharina Bilotti, John M. Pryor, Eric J. Cantor, Barry Canton, Thomas F. Knight, Thomas C. Evans Jr., Gregory J. S. Lohman
bioRxiv 322297; doi: https://doi.org/10.1101/322297

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