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

A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs DOI Creative Commons
Michel Brück,

Tania S. Köbel,

Sophie Dittmar

et al.

iScience, Journal Year: 2024, Volume and Issue: 27(9), P. 110774 - 110774

Published: Aug. 23, 2024

Highlights•A library-based approach for synthetic sRNAs with varying seed region length (SRL)•Synthetic RybB are processed by RNase E at AU-rich sequence motifs•Accessibility of SgrS regions is an important determinant functionality•Importance Hfq and SRL regulatory efficiency depends on the sRNA scaffoldSummaryAll organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) aspect regulation network underlying adaptation such base-pair their target mRNAs, allowing rapid modulation proteome. This post-transcriptional usually facilitated RNA chaperones, as Hfq. have a potential regulators that can be modulated rational design. this study, we use oxacillin susceptibility assays investigate importance based scaffolds in Escherichia coli. presence show 12 nucleotides sufficient regulation. Furthermore, observe scaffold-specific Hfq-dependency processing E. Our results provide information design considerations basic applied research.Graphical abstract

Language: Английский

Citations

2
A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs DOI Creative Commons
Michel Brück,

Tania S. Köbel,

Sophie Dittmar

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 24, 2024

Abstract All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying adaptation such sRNAs base-pair with their target mRNAs, allowing rapid modulation proteome. This post-transcriptional is usually facilitated by RNA chaperones, as Hfq. have a potential synthetic regulators that can be modulated rational design. this study, we use library-based approach and oxacillin susceptibility assays investigate importance seed region length for based on RybB SgrS scaffolds in Escherichia coli . presence Hfq show 12 nucleotides sufficient regulation. Furthermore, observe scaffold-specific Hfq-dependency processing RNase E. Our results provide information design considerations basic applied research.

Language: Английский

Citations

1
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.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 22, 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 four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three 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 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
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