Data from Highly Efficient Single-Pot Scarless Golden Gate Assembly

Dataset · Tue Apr 23 00:00:00 EDT 2019

DOI:https://doi.org/10.13012/B2IDB-1448896_V1· OSTI ID:3013649

HamediRad, Mohammad ^[1]; Weisberg, Scott ^[2]; Chao, Ran ^[1]; Lian, Jiazhang ^[3]; Zhao, Huimin ^[4]

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1. Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Institute for Genomic Biology; Department of Chemical and Biomolecular Engineering, Univ. of Illinois Urbana-Champaign, IL, USA
2. Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Institute for Genomic Biology; Department of Biochemistry, Univ. of Illinois at Urbana-Champaign, IL, USA
3. Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Institute for Genomic Biology; Department of Chemical and Biomolecular Engineering, Univ. of Illinois Urbana-Champaign, IL, USA; College of Chemical and Biological Engineering, Univ. of Illinois Urbana-Champaign, IL, USA
4. Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Institute for Genomic Biology; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); Department of Chemical and Biomolecular Engineering, Univ. of Illinois Urbana-Champaign, IL, USA; Department of Biochemistry, Univ. of Illinois Urbana-Champaign, IL, USA; Department of Chemistry, Univ. of Illinois Urbana-Champaign, IL, USA; Department of Bioengineering, Univ. of Illinois Urbana-Champaign, IL, USA

Golden Gate assembly is one of the most widely used DNA assembly methods due to its robustness and modularity. However, despite its popularity, the need for BsaI-free parts, the introduction of scars between junctions, as well as the lack of a comprehensive study on the linkers hinders its more widespread use. Here, we first developed a novel sequencing scheme to test the efficiency and specificity of 96 linkers of 4-bp length and experimentally verified these linkers and their effects on Golden Gate assembly efficiency and specificity. We then used this sequencing data to generate 200 distinct linker sets that can be used by the community to perform efficient Golden Gate assemblies of different sizes and complexity. We also present a single-pot scarless Golden Gate assembly and BsaI removal scheme and its accompanying assembly design software to perform point mutations and Golden Gate assembly. This assembly scheme enables scarless assembly without compromising efficiency by choosing optimized linkers near assembly junctions.

• Research Organization: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); University of Illinois Urbana-Champaign
• Sponsoring Organization: U.S. Department of Energy (DOE)
• DOE Contract Number: SC0018420
• OSTI ID: 3013649
• Country of Publication: United States
• Language: English

Related Subjects

Conversion
Genome Engineering
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