In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly DOI Creative Commons
Stijn T. de Vries,

Tania S. Köbel,

Ahmet Sanal

et al.

Synthetic Biology, Journal Year: 2024, Volume and Issue: 9(1)

Published: Jan. 1, 2024

Abstract Golden Gate cloning has become one of the most important DNA assembly strategies. The construction standardized and reusable part libraries, their into transcription units, subsequent multigene constructs is highly reliable sustainable. Researchers can quickly construct derivatives assemblies or entire pathways, importantly, standardization compatible with laboratory automation. Most strategies rely on 4-nt overhangs generated by commonly used Type IIS enzymes. However, reduction to 3-nt allows use codons as fusion sites reduces potential scar sequences. This particularly when studying biological functions, additional nucleotides may alter structure stability transcribed RNA. To address this issue we SapI, a enzyme generating three nucleotide overhangs, for unit assembly, allowing codon-based in coding We created corresponding plasmid toolbox basic generation workflow term In-Cloning. In-Cloning downstream Modular Cloning standard developed Sylvestre Marillonnet’s group constructs. plasmids not be model organism choice. Therefore, have called Out-Cloning rapidly generate acceptor plasmids. uses parts that are assembled using flexible linkers. systematic needed transfer interest.

Language: Английский

Advancements in Golden Gate Cloning: A Comprehensive Review DOI

Jesús Laborda-Mansilla,

Eva García-Ruiz

Methods in molecular biology, Journal Year: 2024, Volume and Issue: unknown, P. 481 - 500

Published: Sept. 17, 2024

Language: Английский

Citations

0
In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly DOI Creative Commons
Stijn T. de Vries,

Tania S. Köbel,

Ahmet Sanal

et al.

Synthetic Biology, Journal Year: 2024, Volume and Issue: 9(1)

Published: Jan. 1, 2024

Abstract Golden Gate cloning has become one of the most important DNA assembly strategies. The construction standardized and reusable part libraries, their into transcription units, subsequent multigene constructs is highly reliable sustainable. Researchers can quickly construct derivatives assemblies or entire pathways, importantly, standardization compatible with laboratory automation. Most strategies rely on 4-nt overhangs generated by commonly used Type IIS enzymes. However, reduction to 3-nt allows use codons as fusion sites reduces potential scar sequences. This particularly when studying biological functions, additional nucleotides may alter structure stability transcribed RNA. To address this issue we SapI, a enzyme generating three nucleotide overhangs, for unit assembly, allowing codon-based in coding We created corresponding plasmid toolbox basic generation workflow term In-Cloning. In-Cloning downstream Modular Cloning standard developed Sylvestre Marillonnet’s group constructs. plasmids not be model organism choice. Therefore, have called Out-Cloning rapidly generate acceptor plasmids. uses parts that are assembled using flexible linkers. systematic needed transfer interest.

Language: Английский

Citations

0