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

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing DOI
Adán Andrés Ramírez Rojas, Cedric K. Brinkmann, Daniel Schindler

et al.

Methods in molecular biology, Journal Year: 2024, Volume and Issue: unknown, P. 171 - 196

Published: Sept. 17, 2024

Language: Английский

Citations

4
Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly DOI
Stijn T. de Vries,

Laura Kley,

Daniel Schindler

et al.

Methods in molecular biology, Journal Year: 2024, Volume and Issue: unknown, P. 105 - 131

Published: Sept. 17, 2024

Language: Английский

Citations

3
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
Rationally designed chromosome fusion does not prevent rapid growth of Vibrio natriegens DOI Creative Commons
Lea Ramming, Daniel Stukenberg, María del Carmen Sánchez Olmos

et al.

Communications Biology, Journal Year: 2024, Volume and Issue: 7(1)

Published: May 2, 2024

DNA replication is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin replication, with proceeding at about 1000 bp per second. For model organism, Escherichia coli, this translates into time 40 min its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping cycles maximum short doubling 20 min. The fastest growing bacterium known, Vibrio natriegens, able to replicate generation less than 10 It has bipartite genome chromosome sizes 3.2 and 1.9 Mb. Is simultaneous two origins prerequisite rapid growth? We fused V. natriegens create strain carrying one replication. Compared parental, showed no significant deviation in growth rate. This suggests that split not growth.

Language: Английский

Citations

1
Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications DOI Creative Commons
Nadiia Pozhydaieva, Franziska Anna Billau, Maik Wolfram-Schauerte

et al.

PLoS Genetics, Journal Year: 2024, Volume and Issue: 20(9), P. e1011384 - e1011384

Published: Sept. 4, 2024

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer way to explore these via site-specific mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET temporarily reduce modifications, facilitating Cas nuclease enhancing This approach enables precise targeting seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for infection. Furthermore, temporally removing we elucidated effects on T4 infections without necessitating gene deletions. results present strategy enabling investigation epigenome functions streamlining engineering with modifications. The described temporal modulation valuable synthetic biology fundamental research comprehend generation mutants.

Language: Английский

Citations

1
LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning DOI
Yueming Long, Ariane Mora, Francesca-Zhoufan Li

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 14(1), P. 230 - 238

Published: Dec. 24, 2024

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function for entire protein-coding genes. LevSeq integrates into existing engineering workflows and comes open-source software analysis visualization. The facilitates data-driven by consolidating inform provide requisite machine learning-guided (MLPE). enables quality control of mutagenesis libraries prior screening, which reduces time resource costs. Simulation studies demonstrate LevSeq's ability accurately detect variants under various experimental conditions. Finally, show utility in protoglobins new-to-nature chemistry. Widespread adoption sharing will enhance our understanding landscapes empower evolution.

Language: Английский

Citations

1
T4 phage RNA is NAD-capped and alters the NAD-cap epitranscriptome of Escherichia coli during infection through a phage-encoded decapping enzyme DOI Creative Commons
Maik Wolfram-Schauerte, Anastassiya Moskalchuk, Nadiia Pozhydaieva

et al.

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

Published: April 4, 2024

ABSTRACT Nicotinamide adenine dinucleotide (NAD) serves as a cap-like structure on cellular RNAs (NAD-RNAs) in all domains of life including the bacterium Escherichia coli . NAD also acts key molecule phage-host interactions, where bacterial immune systems deplete to abort phage infection. Nevertheless, NAD-RNAs have not yet been identified during infections bacteria and mechanisms their synthesis degradation are unknown this context. The T4 that specifically infects E. presents an important model study infections, but systematic analysis presence dynamics infection is lacking. Here, we investigate dual manner. By applying time-resolved captureSeq, identify NAD-capped host transcripts dynamic regulation We provide evidence – reported earlier generated by RNA polymerase initiating transcription with at canonical start sites. In addition, characterize NudE.1 phage-encoded Nudix hydrolase first NAD-RNA decapping enzyme. phages carrying inactive display delayed lysis phenotype. This investigates for time epitranscriptome its host, thereby introducing epitranscriptomics field research.

Language: Английский

Citations

0
Pacybara: accurate long-read sequencing for barcoded mutagenized allelic libraries DOI Creative Commons
Jochen Weile,

Gabrielle Ferra,

Gabriel Boyle

et al.

Bioinformatics, Journal Year: 2024, Volume and Issue: 40(4)

Published: March 29, 2024

Long-read sequencing technologies, an attractive solution for many applications, often suffer from higher error rates. Alignment of multiple reads can improve base-calling accuracy, but some e.g. mutagenized libraries where distinct clones differ by one or few variants, require the use barcodes unique molecular identifiers. Unfortunately, errors interfere with correct barcode identification, and a given sequence may be linked to independent within library.

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.

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