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Rapid 40 kb genome construction from 52 parts

View ORCID ProfileJohn M. Pryor, View ORCID ProfileVladimir Potapov, View ORCID ProfileNilisha Pokhrel, View ORCID ProfileGregory J. S. Lohman
doi: https://doi.org/10.1101/2020.12.22.424019
John M. Pryor
Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Vladimir Potapov
Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Nilisha Pokhrel
Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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Gregory J. S. Lohman
Research Department, New England Biolabs, Ipswich, MA, 01938, USA
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  • For correspondence: lohman@neb.com
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ABSTRACT

Large DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found that optimized assembly permitted >50 DNA fragments to be faithfully assembled in a single reaction. We applied these insights to genome construction, carrying out rapid assembly of the 40 kb T7 bacteriophage genome from 52 parts and recovering infectious phage particles after cellular transformation. The new Golden Gate assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Competing Interest Statement

The authors are employees of New England Biolabs, manufacturer of reagents described in the manuscript.

Copyright 
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 December 22, 2020.
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Rapid 40 kb genome construction from 52 parts
John M. Pryor, Vladimir Potapov, Nilisha Pokhrel, Gregory J. S. Lohman
bioRxiv 2020.12.22.424019; doi: https://doi.org/10.1101/2020.12.22.424019
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Rapid 40 kb genome construction from 52 parts
John M. Pryor, Vladimir Potapov, Nilisha Pokhrel, Gregory J. S. Lohman
bioRxiv 2020.12.22.424019; doi: https://doi.org/10.1101/2020.12.22.424019

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