{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T21:07:06Z","timestamp":1762981626996,"version":"3.41.2"},"reference-count":21,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2023,5,19]],"date-time":"2023-05-19T00:00:00Z","timestamp":1684454400000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2023,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>A gene deletion by allelic exchange via homologous recombination from a bacterial genome represents a valuable genetic tool for studying a role(s) of determinants involved in various aspects of pathogenesis. Due to chlamydial obligate intracellular lifestyle and comparatively low transformation rate, the mutagenesis of <jats:italic>Chlamydia<\/jats:italic> utilizes types of suicide vectors that have to be maintained and propagated by the bacteria throughout several rounds of their intracellular developmental cycle. These deletion constructs must be lost by chlamydiae once null mutant formation is achieved. pKW is a small, 5.45 bp, pUC19\u2010derived vector, which has been recently successfully employed for the generation of deletion mutants in <jats:italic>C. trachomatis<\/jats:italic>, serovariant D, and <jats:italic>C. muridarum<\/jats:italic>. This vector contains both, <jats:italic>E. coli<\/jats:italic> as well as chlamydial species\u2010specific plasmid origins of replication, allowing for its propagation by both bacterial genera under a selective pressure. However, once the selective antibiotic is removed from culture, chlamydiae rapidly lose pKW, and the subsequent reintroduction of the selective antibiotic back to chlamydiae\u2010infected cells results efficiently in the selection of generated deletion mutants. Protocols provided here describe in detail the preparation of pKW deletion constructs for <jats:italic>C. trachomatis<\/jats:italic> and <jats:italic>C. muridarum<\/jats:italic> applicable for chlamydial transformation and the production of null mutant in non\u2010essential genes. Protocols provided here, are describing in detail methods for assembly of the pKW shuttle vector and generation of deletion mutants in <jats:italic>C. trachomatis<\/jats:italic> and <jats:italic>C. muridarum<\/jats:italic>. \u00a9 2023 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Assembly of pKW shuttle vector<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Generation of a deletion mutant in <jats:italic>C. trachomatis<\/jats:italic>, serovars D and L2 and, <jats:italic>Chlamydia muridarum<\/jats:italic><\/jats:p><jats:p><jats:bold>Support Protocol<\/jats:bold>: Transformation of <jats:italic>C. trachomatis<\/jats:italic>, serovars B<\/jats:p>","DOI":"10.1002\/cpz1.775","type":"journal-article","created":{"date-parts":[[2023,5,19]],"date-time":"2023-05-19T12:54:20Z","timestamp":1684500860000},"source":"Crossref","is-referenced-by-count":2,"title":["Transformation and Mutagenesis of <i>Chlamydia trachomatis<\/i> and <i>C. muridarum<\/i> Utilizing pKW Vector"],"prefix":"10.1002","volume":"3","author":[{"given":"Katerina","family":"Wolf","sequence":"first","affiliation":[{"name":"Department of Microbiology, Immunology &amp; Molecular Genetics University of Kentucky College of Medicine  Lexington Kentucky"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2023,5,19]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.1.5.434\u2010439.1975"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0378\u20101135(00)00213\u20103"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/iai.00453\u201022"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00841\u201019"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1318"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0703218104"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00479\u201018"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.8.4.451"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010019\u201054886\u20105"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.mi.20.100166.000543"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01817\u201015"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/0147\u2010619X(86)90079\u2010X"},{"issue":"11","key":"e_1_2_8_14_1","first-page":"844","article-title":"Global data on visual impairment in the year 2002","volume":"82","author":"Resnikoff S.","year":"2004","journal-title":"Bulletin of the World Health Organization"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.syapm.2013.12.004"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.12688\/wellcomeopenres.13905.1"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.282.5389.754"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/0147\u2010619X(92)90025\u20106"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1099\/00221287\u2010143\u20106\u20101847"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002258"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u20104939\u20109694\u20100_12"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1865\u20101682.2011.01274.x"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.775","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,14]],"date-time":"2025-06-14T10:12:15Z","timestamp":1749895935000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.775"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5]]},"references-count":21,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2023,5]]}},"alternative-id":["10.1002\/cpz1.775"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.775","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"type":"print","value":"2691-1299"},{"type":"electronic","value":"2691-1299"}],"subject":[],"published":{"date-parts":[[2023,5]]},"article-number":"e775"}}