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Title: Highly Efficient Single-Pot Scarless Golden Gate Assembly

Abstract

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.

Authors:
 [1];  [1];  [1]; ORCiD logo [1]; ORCiD logo [2]
  1. Zhejiang Univ., Hangzhou (China)
  2. Zhejiang Univ., Hangzhou (China); Univ. of Illinois at Urbana−Champaign, Urbana, IL (United States)
Publication Date:
Research Org.:
Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1542926
Grant/Contract Number:  
SC0018420
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 8; Journal Issue: 5; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA assembly; Golden Gate assembly; synthetic biology

Citation Formats

HamediRad, Mohammad, Weisberg, Scott, Chao, Ran, Lian, Jiazhang, and Zhao, Huimin. Highly Efficient Single-Pot Scarless Golden Gate Assembly. United States: N. p., 2019. Web. doi:10.1021/acssynbio.8b00480.
HamediRad, Mohammad, Weisberg, Scott, Chao, Ran, Lian, Jiazhang, & Zhao, Huimin. Highly Efficient Single-Pot Scarless Golden Gate Assembly. United States. https://doi.org/10.1021/acssynbio.8b00480
HamediRad, Mohammad, Weisberg, Scott, Chao, Ran, Lian, Jiazhang, and Zhao, Huimin. Tue . "Highly Efficient Single-Pot Scarless Golden Gate Assembly". United States. https://doi.org/10.1021/acssynbio.8b00480. https://www.osti.gov/servlets/purl/1542926.
@article{osti_1542926,
title = {Highly Efficient Single-Pot Scarless Golden Gate Assembly},
author = {HamediRad, Mohammad and Weisberg, Scott and Chao, Ran and Lian, Jiazhang and Zhao, Huimin},
abstractNote = {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.},
doi = {10.1021/acssynbio.8b00480},
journal = {ACS Synthetic Biology},
number = 5,
volume = 8,
place = {United States},
year = {2019},
month = {4}
}

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Works referencing / citing this record:

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journal, July 2019


Towards a fully automated algorithm driven platform for biosystems design
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