{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T12:09:18Z","timestamp":1790251758909,"version":"4.1.0"},"reference-count":38,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["MicrobiologyOpen"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Protoplast transformation has been an effective method for introducing plasmid DNA into\n                    <jats:italic>Bacillus<\/jats:italic>\n                    species; however, its application to deliver complete bacteriophage genomes has not been demonstrated previously. Here, we describe an optimized and reproducible method for forming\n                    <jats:italic>Geobacillus stearothermophilus<\/jats:italic>\n                    protoplasts and efficiently transfecting them with purified recombinant or wild\u2010type genomic DNA from the thermophilic bacteriophage TP\u201084. Systematic optimization of key parameters, including bacterial cell density, lysozyme concentration, polyethylene glycol type and concentration, and DNA input, enabled robust phage genomic DNA uptake and subsequent production of infectious phage particles. In addition, we optimized and scaled up a single\u2010layer solid\u2010state bacteriophage propagation procedure for integration into the TP\u201084 genome\u2010engineering workflow, enabling the recovery of high\u2010titer lysates and sufficient genomic DNA for sequencing and subsequent genome manipulation from a single Petri dish. In our experimental system, solid\u2010state propagation also provided greater stability of recombinant phage variants than propagation in liquid bacterial cultures. Together, these methods establish an integrated protoplast\u2010based system for phage genomic DNA delivery, rebooting, propagation, and recovery, enabling controlled manipulation of bacteriophage genomes and facilitating the rapid generation and analysis of recombinant variants.\n                  <\/jats:p>","DOI":"10.1002\/mbo3.70421","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T11:39:45Z","timestamp":1790249985000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An Integrated System for\n                    <i>Geobacillus<\/i>\n                    Protoplast Formation, Transfection, Recombinant Genomes Rebooting, Efficient Propagation, and Genomic DNA Isolation of the Thermophilic Bacteriophage TP\u201084"],"prefix":"10.1002","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9510-2557","authenticated-orcid":false,"given":"Ireneusz","family":"Sobolewski","sequence":"first","affiliation":[{"name":"Department of Molecular Biotechnology, Faculty of Chemistry University of Gdansk Gdansk Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3972-5450","authenticated-orcid":false,"given":"Katarzyna","family":"Adamowicz","sequence":"additional","affiliation":[{"name":"Department of Molecular Biotechnology, Faculty of Chemistry University of Gdansk Gdansk Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-6067-313X","authenticated-orcid":false,"given":"Jan","family":"Chodorski","sequence":"additional","affiliation":[{"name":"Department of Molecular Biotechnology, Faculty of Chemistry University of Gdansk Gdansk Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1345-3271","authenticated-orcid":false,"given":"Piotr M.","family":"Skowron","sequence":"additional","affiliation":[{"name":"Department of Molecular Biotechnology, Faculty of Chemistry University of Gdansk Gdansk Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2479-3634","authenticated-orcid":false,"given":"Agnieszka","family":"Zylicz\u2010Stachula","sequence":"additional","affiliation":[{"name":"Department of Molecular Biotechnology, Faculty of Chemistry University of Gdansk Gdansk Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-006-0849-1"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.syapm.2016.09.004"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.syapm.2018.07.001"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-014-5746-z"},{"key":"e_1_2_10_6_1","volume-title":"The Bacteriophages","author":"Calendar 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