Ligation independent cloning: an efficient tool for high-throughput cloning and modular cDNA assembly

AbstractCovalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.

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pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

Microbiology ◽

10.1099/mic.0.035758-0 ◽

2010 ◽

Vol 156 (6) ◽

pp. 1600-1608 ◽

Cited By ~ 47

Author(s):

Eric Botella ◽

Mark Fogg ◽

Matthieu Jules ◽

Sjouke Piersma ◽

Geoff Doherty ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

High Throughput ◽

Chromosomal Locus ◽

High Throughput Analysis ◽

Throughput Analysis ◽

Ligation Independent Cloning ◽

Phosphate Availability ◽

Integrative Plasmid ◽

Versatile Tool

Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis.

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High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PLoS ONE ◽

10.1371/journal.pone.0018900 ◽

2011 ◽

Vol 6 (4) ◽

pp. e18900 ◽

Cited By ~ 10

Author(s):

Hakan Dortay ◽

Usha Madhuri Akula ◽

Christin Westphal ◽

Marie Sittig ◽

Bernd Mueller-Roeber

Keyword(s):

Protein Expression ◽

High Throughput ◽

Fluorescent Protein ◽

Ligation Independent Cloning ◽

Infrared Fluorescent Protein

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Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects

Frontiers in Microbiology ◽

10.3389/fmicb.2021.635227 ◽

2021 ◽

Vol 12 ◽

Author(s):

Riyu Zhang ◽

Wensheng Xu ◽

Shuai Shao ◽

Qiyao Wang

Keyword(s):

High Throughput ◽

Genetic Screening ◽

Underlying Mechanism ◽

Biological Processes ◽

Efficient Tool ◽

Guide Rna ◽

Crispr Interference ◽

Repeat Sequences ◽

A Genome ◽

Short Palindromic Repeat

Functional genetic screening is an important method that has been widely used to explore the biological processes and functional annotation of genetic elements. CRISPR/Cas (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein) is the newest tool in the geneticist’s toolbox, allowing researchers to edit a genome with unprecedented ease, accuracy, and high-throughput. Most recently, CRISPR interference (CRISPRi) has been developed as an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single-guide RNA (sgRNA) to repress sequence-specific genes. In this review, we summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific. Moreover, we demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis. The recent development of the CRISPRi system will provide a high-throughput, practical, and efficient tool for the discovery of functionally important genes in bacteria.

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