{"id":38427,"date":"2021-06-07T14:20:19","date_gmt":"2021-06-07T14:20:19","guid":{"rendered":"https:\/\/matis.is\/?p=38427"},"modified":"2021-06-07T14:20:20","modified_gmt":"2021-06-07T14:20:20","slug":"generation-of-targeted-deletions-in-the-genome-of-rhodothermus-marinus","status":"publish","type":"post","link":"https:\/\/matis.is\/en\/greinar\/generation-of-targeted-deletions-in-the-genome-of-rhodothermus-marinus\/","title":{"rendered":"Generation of targeted deletions in the genome of Rhodothermus marinus"},"content":{"rendered":"<p>The aim of this work was to develop an approach for chromosomal engineering of the thermophile\u00a0<em>Rhodothermus marinus<\/em>. A selection strategy for\u00a0<em>R. marinus<\/em>\u00a0had previously been developed; this strategy was based on complementing a restriction-negative\u00a0<em>trpB<\/em>\u00a0strain with the\u00a0<em>R. marinustrpB<\/em>\u00a0gene. The current work identified an additional selective marker,\u00a0<em>purA<\/em>, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp<sup>+<\/sup>\u00a0selection was used during the deletion of\u00a0<em>purA<\/em>\u00a0from the\u00a0<em>R. marinus<\/em>\u00a0chromosomes. The alternative Ade<sup>+<\/sup>\u00a0selection was in turn used while deleting the endogenous\u00a0<em>trpB<\/em>\u00a0gene. Since both deletions are unmarked, the\u00a0<em>purA<\/em>\u00a0and\u00a0<em>trpB<\/em>\u00a0markers may be reused. Through the double deletant SB-62 (\u0394<em>trpB<\/em>\u00a0\u0394<em>purA<\/em>), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in\u00a0<em>R. marinus<\/em>\u00a0strain SB-62 (\u0394<em>trpB<\/em>\u00a0\u0394<em>purA<\/em>) was demonstrated by targeting putative carotenoid biosynthesis genes,\u00a0<em>crtBI<\/em>, using a linear molecule containing a marked deletion with 717 and 810 bp of 5 \u2032 and 3 \u2032 homologous sequences, respectively. The resulting Trp<sup>+<\/sup>\u00a0transformants were colorless rather than orange-red. The correct replacement of an internal\u00a0<em>crtBI<\/em>\u00a0fragment with the\u00a0<em>trpB<\/em>\u00a0marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the\u00a0<em>R. marinus<\/em>\u00a0chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.<\/p>\n\n\n\n<p><strong><a href=\"https:\/\/journals.asm.org\/doi\/10.1128\/AEM.02070-10\">Link to article<\/a><\/strong><\/p>","protected":false},"excerpt":{"rendered":"<p>The aim of this work was to develop an approach for chromosomal engineering of the thermophile\u00a0Rhodothermus marinus. A selection strategy [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"template-greinar.php","format":"standard","meta":{"_acf_changed":false,"_uag_custom_page_level_css":"","site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"default","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","ast-disable-related-posts":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"footnotes":""},"categories":[2011],"tags":[],"class_list":["post-38427","post","type-post","status-publish","format-standard","hentry","category-greinar"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.4 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Generation of targeted deletions in the genome of\u00a0Rhodothermus marinus - Mat\u00eds<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/matis.is\/en\/greinar\/generation-of-targeted-deletions-in-the-genome-of-rhodothermus-marinus\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Generation of targeted deletions in the genome of\u00a0Rhodothermus marinus - Mat\u00eds\" \/>\n<meta property=\"og:description\" content=\"The aim of this work was to develop an approach for chromosomal engineering of the thermophile\u00a0Rhodothermus marinus. 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