Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay
| Autor: | Kasurinen, Jutta, Spruit, Cindy M., Wicklund, Anu, Pajunen, Maria I., Skurnik, Mikael |
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| Přispěvatelé: | HUMI - Human Microbiome Research, Department of Bacteriology and Immunology, Medicum, Faculty of Medicine, University of Helsinki, Research Programs Unit, HUSLAB, Glycoscience Group, Biosciences, Helsinki One Health (HOH), Mikael Skurnik / Principal Investigator, Helsinki University Hospital Area |
| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular Proteomics toxic protein screen Protein Conformation GENOMES PHAGE Genome Viral Microbiology Host Specificity Article Structure-Activity Relationship Viral Proteins bacteriophage SEARCH RECG Humans Bacteriophages Amino Acid Sequence HELICASE Toxins Biological 11832 Microbiology and virology IDENTIFICATION Computational Biology High-Throughput Nucleotide Sequencing Genomics Sequence Analysis DNA hypothetical proteins of unknown function QR1-502 ESCHERICHIA-COLI DISCOVERY COMPLEXES next-generation sequencing 3111 Biomedicine |
| Zdroj: | Viruses Viruses, Vol 13, Iss 750, p 750 (2021) Volume 13 Issue 5 |
| ISSN: | 1999-4915 |
| Popis: | Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules. |
| Databáze: | OpenAIRE |
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