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.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1021/acssynbio.8b00480 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Correction to: Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.
Methods Mol Biol
January 2025
Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in .
ACS Synth Biol
January 2025
Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, U.K.
Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts.
View Article and Find Full Text PDFA point mutation in the pepper veinal mottle virus 6K1 protein yields a stable attenuated strain for engineering virus resistance in pepper plants.
Plant Dis
January 2025
No. 58, Renmin AvenueHaikou, China, 570228;
A total of 164 viruses have been identified in peppers worldwide. To combat viruses, pathogen-derived resistance (PDR) has been employed by expressing a viral genomic segment or a viral protein in host plants. Unfortunately, peppers are recalcitrant to genetic transformation and regeneration.
View Article and Find Full Text PDFFramework for developing self-actualization skills in young adults with autism.
Health Care Transit
January 2024
Emeritus, UCSF, School of Nursing, Family Health Care Nursing, 2 Koret Way, N411Y, San Francisco, CA 94143-0606, USA.
Purpose: Young adults with autism spectrum disorder (YAASD) often lack the skills needed for successful adult living and subsequently develop lower quality of life and health in adulthood. Therefore, we developed a framework to support resilience and improve quality of life in YAASD, which will be used to develop a life-skills curriculum for YAASD.
Methods: We conducted a qualitative study of YAASD, parents of YAASD and service providers recruited from agencies that serve YAASD using Grounded Theory.
PHARMACOKINETICS OF CEFTAZIDIME IN SIGNAL CRAYFISH () FOLLOWING A SINGLE DOSE.
J Zoo Wildl Med
December 2024
Department of Medicine & Epidemiology, Davis, CA 95616, USA,
Crustaceans are housed in zoos and aquariums and have also gained importance in the private sector and food industry. Shell lesions are common and often attributed to bacterial infections. However, few controlled studies have been performed evaluating antibiotics in crustaceans.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!