An efficient, scarless, selection-free technology for phage engineering
Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes....
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Taylor & Francis Group
2023-12-01
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| Series: | RNA Biology |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2023.2270344 |
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| author | Moran G. Goren Tridib Mahata Udi Qimron |
| author_facet | Moran G. Goren Tridib Mahata Udi Qimron |
| author_sort | Moran G. Goren |
| collection | DOAJ |
| description | Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1–14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50–201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text] |
| format | Article |
| id | doaj-art-a6fdfea4e56b4e2ba8afee32b82fd626 |
| institution | OA Journals |
| issn | 1547-6286 1555-8584 |
| language | English |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj-art-a6fdfea4e56b4e2ba8afee32b82fd6262025-08-20T02:17:00ZengTaylor & Francis GroupRNA Biology1547-62861555-85842023-12-0120183083510.1080/15476286.2023.2270344An efficient, scarless, selection-free technology for phage engineeringMoran G. Goren0Tridib Mahata1Udi Qimron2Department of Clinical Microbiology and Immunology, School of Medicine, Tel Aviv University, Tel Aviv, IsraelDepartment of Clinical Microbiology and Immunology, School of Medicine, Tel Aviv University, Tel Aviv, IsraelDepartment of Clinical Microbiology and Immunology, School of Medicine, Tel Aviv University, Tel Aviv, IsraelMost recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1–14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50–201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text]https://www.tandfonline.com/doi/10.1080/15476286.2023.2270344DNA engineeringBacteriophagespORTMAGE |
| spellingShingle | Moran G. Goren Tridib Mahata Udi Qimron An efficient, scarless, selection-free technology for phage engineering RNA Biology DNA engineering Bacteriophages pORTMAGE |
| title | An efficient, scarless, selection-free technology for phage engineering |
| title_full | An efficient, scarless, selection-free technology for phage engineering |
| title_fullStr | An efficient, scarless, selection-free technology for phage engineering |
| title_full_unstemmed | An efficient, scarless, selection-free technology for phage engineering |
| title_short | An efficient, scarless, selection-free technology for phage engineering |
| title_sort | efficient scarless selection free technology for phage engineering |
| topic | DNA engineering Bacteriophages pORTMAGE |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2023.2270344 |
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