Highly Efficient Single-Pot Scarless Golden Gate Assembly

HamediRad, Mohammad; Weisberg, Scott; Chao, Ran; Lian, Jiazhang; Zhao, Huimin

doi:10.1021/acssynbio.8b00480

Highly Efficient Single-Pot Scarless Golden Gate Assembly

Journal Article · Tue Apr 23 00:00:00 EDT 2019 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.8b00480· OSTI ID:1542926

HamediRad, Mohammad ^[1]; Weisberg, Scott ^[2]; Chao, Ran ^[2]; ^[2]; ^[3]

Zhejiang Univ., Hangzhou (China); CABBI
Zhejiang Univ., Hangzhou (China)
Zhejiang Univ., Hangzhou (China); Univ. of Illinois at Urbana−Champaign, Urbana, IL (United States)

We report that 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. Finally, this assembly scheme enables scarless assembly without compromising efficiency by choosing optimized linkers near assembly junctions.

View Accepted Manuscript (DOE)

Research Organization:: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: SC0018420

OSTI ID:: 1542926

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 5 Vol. 8; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation Chockalingam, Karuppiah; Peng, Zeyu; Vuong, Christine N. Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-020-59745-2	journal	February 2020
Towards a fully automated algorithm driven platform for biosystems design HamediRad, Mohammad; Chao, Ran; Weisberg, Scott Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-13189-z	journal	November 2019
Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design Püllmann, Pascal; Ulpinnis, Chris; Marillonnet, Sylvestre Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-47376-1	journal	July 2019

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