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Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated <jats:italic>Pseudomonas aeruginosa<\/jats:italic> phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a <jats:italic>Galleria mellonella<\/jats:italic> infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of <jats:italic>P. aeruginosa<\/jats:italic> infections.<\/jats:p>","DOI":"10.1038\/s41598-021-81580-2","type":"journal-article","created":{"date-parts":[[2021,1,25]],"date-time":"2021-01-25T11:06:41Z","timestamp":1611572801000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":62,"title":["Designing P. aeruginosa synthetic phages with reduced genomes"],"prefix":"10.1038","volume":"11","author":[{"given":"Diana P.","family":"Pires","sequence":"first","affiliation":[]},{"given":"Rodrigo","family":"Monteiro","sequence":"additional","affiliation":[]},{"given":"Dalila","family":"Mil-Homens","sequence":"additional","affiliation":[]},{"given":"Ars\u00e9nio","family":"Fialho","sequence":"additional","affiliation":[]},{"given":"Timothy K.","family":"Lu","sequence":"additional","affiliation":[]},{"given":"Joana","family":"Azeredo","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2021,1,25]]},"reference":[{"key":"81580_CR1","doi-asserted-by":"publisher","first-page":"1432","DOI":"10.1038\/s41564-019-0503-9","volume":"4","author":"S Hernando-Amado","year":"2019","unstructured":"Hernando-Amado, S., Coque, T. 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