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Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of\n              <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n              . As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of\n              <jats:italic>P. aeruginosa<\/jats:italic>\n              . We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered\n              <jats:italic>P. aeruginosa<\/jats:italic>\n              phages.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>\n                <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant\n                <jats:italic>P. aeruginosa<\/jats:italic>\n                tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.\n              <\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.02897-23","type":"journal-article","created":{"date-parts":[[2024,1,31]],"date-time":"2024-01-31T14:00:23Z","timestamp":1706709623000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":22,"title":["A synthetic biology approach to assemble and reboot clinically relevant\n            <i>Pseudomonas aeruginosa<\/i>\n            tailed phages"],"prefix":"10.1128","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6266-9381","authenticated-orcid":false,"given":"Thomas","family":"Ipoutcha","sequence":"first","affiliation":[{"name":"Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6471-9434","authenticated-orcid":false,"given":"Ratanachat","family":"Racharaks","sequence":"additional","affiliation":[{"name":"Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-0368-0434","authenticated-orcid":false,"given":"Stefanie","family":"Huttelmaier","sequence":"additional","affiliation":[{"name":"Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6276-7980","authenticated-orcid":false,"given":"Cole J.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7131-3691","authenticated-orcid":false,"given":"Egon A.","family":"Ozer","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0966-2014","authenticated-orcid":false,"given":"Erica M.","family":"Hartmann","sequence":"additional","affiliation":[{"name":"Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois, USA"},{"name":"Center for Synthetic Biology, Northwestern University, Evanston, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.7573\/dic.212527"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(22)02185-7"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1007\/s12223-022-00990-5"},{"key":"e_1_3_4_5_2","unstructured":"ECSF. 2020. 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