{"id":1334,"date":"2018-11-07T22:06:37","date_gmt":"2018-11-07T22:06:37","guid":{"rendered":"https:\/\/wp.uthscsa.edu\/biochemistry\/?page_id=1334"},"modified":"2021-01-22T09:49:31","modified_gmt":"2021-01-22T15:49:31","slug":"growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media","status":"publish","type":"page","link":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/","title":{"rendered":"Growth of Prototrophic E. coli Strains in D2O Minimal Salts Media"},"content":{"rendered":"<p>The following is a protocol for growing 1.0 L of\u00a0E. coli\u00a0in deuterium oxide (D<sub>2<\/sub>O)-M9 salts medium. Due to the slow growth of cells in D<sub>2<\/sub>O minimal medium, the D<sub>2<\/sub>O culture is inoculated with cells at 0.3 A<sub>600<\/sub>\u00a0grown on H<sub>2<\/sub>O -M9 Salts medium.<\/p>\n<p>To prepare 1L of D<sub>2<\/sub>O medium, follow all the 11 steps listed in the Minimal Salts Media protocol, but a) substitute the water with deuterium oxide (D<sub>2<\/sub>O) in all the solutions prepared, and b) do not autoclave D<sub>2<\/sub>O itself, MgSO<sub>4<\/sub>, glucose, CaCl<sub>2<\/sub>, or yeast extract for sterilization, only filter (0.22<span style=\"font-family: Symbol\">m<\/span>M filter). Also if using\u00a0<sup>15<\/sup>N or\u00a0<sup>13<\/sup>C or both, remember to prepare the\u00a0<sup>15<\/sup>NH<sub>4<\/sub>Cl or\u00a0<sup>13<\/sup>C-glucose, respectively in D<sub>2<\/sub>O as well.<\/p>\n<p>The protocol below is for culturing cells on 1.0 L of D<sub>2<\/sub>O medium starting from cells growing on H<sub>2<\/sub>O-M9 salts medium:<\/p>\n<p>1. Inoculate 200mL of H<sub>2<\/sub>O-M9 salts medium from fresh colony (as described in\u00a0<a href=\"http:\/\/nmr.uthscsa.edu\/html\/education\/m9.shtml\">Minimal Salts media<\/a>\u00a0protocol)<\/p>\n<p>2. Let cells rise to an A<sub>600<\/sub>\u00a0of ~0.3.<\/p>\n<p>3. Centrifuge initial 200mL cells in sterile bottles and dump supernatant.<\/p>\n<p>4. Resuspend pellet in 10mL of D<sub>2<\/sub>O-M9 medium (prepared with\u00a0<sup>15<\/sup>N or\u00a0<sup>13<\/sup>C isotope as appropriate).<\/p>\n<p>5. Add the 10mL of resuspended cells to the 1.0 L D<sub>2<\/sub>O culture in sterile flask. This will yield a starting A<sub>600<\/sub>\u00a0of ~ 0.06.<\/p>\n<p>6. Wait for cells to rise at an A<sub>600<\/sub>\u00a0of ~0.7, then induce protein expression with IPTG (or whatever is appropriate) and add 2.5mL of 20% (w\/v) D-glucose (or isotope labeled\u00a0<sup>13<\/sup>C-glucose, as appropriate).<\/p>\n<p>7. After induction is complete (typically 5-7 hours), spin down rest of cells 10 minutes at 7500 r.p.m.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>The following is a protocol for growing 1.0 L of\u00a0E. coli\u00a0in deuterium oxide (D2O)-M9 salts medium. Due to the slow growth of cells in D2O minimal medium, the D2O culture is inoculated with cells at 0.3 A600\u00a0grown on H2O -M9 Salts medium. To prepare 1L of D2O medium, follow all the 11 steps listed in [&hellip;]<\/p>\n","protected":false},"author":161,"featured_media":0,"parent":1008,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-1334","page","type-page","status-publish","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.8 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Growth of Prototrophic E. coli Strains in D2O Minimal Salts Media - Biochemistry<\/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:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Growth of Prototrophic E. coli Strains in D2O Minimal Salts Media - Biochemistry\" \/>\n<meta property=\"og:description\" content=\"The following is a protocol for growing 1.0 L of\u00a0E. coli\u00a0in deuterium oxide (D2O)-M9 salts medium. 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Due to the slow growth of cells in D2O minimal medium, the D2O culture is inoculated with cells at 0.3 A600\u00a0grown on H2O -M9 Salts medium. To prepare 1L of D2O medium, follow all the 11 steps listed in [&hellip;]","og_url":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/","og_site_name":"Biochemistry","article_modified_time":"2021-01-22T15:49:31+00:00","twitter_card":"summary_large_image","twitter_misc":{"Est. reading time":"2 minutes"},"schema":{"@context":"https:\/\/schema.org","@graph":[{"@type":"WebPage","@id":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/","url":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/","name":"Growth of Prototrophic E. coli Strains in D2O Minimal Salts Media - Biochemistry","isPartOf":{"@id":"https:\/\/lsom.uthscsa.edu\/biochemistry\/#website"},"datePublished":"2018-11-07T22:06:37+00:00","dateModified":"2021-01-22T15:49:31+00:00","breadcrumb":{"@id":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/#breadcrumb"},"inLanguage":"en-US","potentialAction":[{"@type":"ReadAction","target":["https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/"]}]},{"@type":"BreadcrumbList","@id":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/growth-of-prototrophic-e-coli-strains-in-d2o-minimal-salts-media\/#breadcrumb","itemListElement":[{"@type":"ListItem","position":1,"name":"Home","item":"https:\/\/lsom.uthscsa.edu\/biochemistry\/"},{"@type":"ListItem","position":2,"name":"Core Facilities","item":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/"},{"@type":"ListItem","position":3,"name":"Biomolecular NMR Core","item":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/"},{"@type":"ListItem","position":4,"name":"Technical Resources","item":"https:\/\/lsom.uthscsa.edu\/biochemistry\/core-facilities\/biomolecular-nmr-core\/technical-resources\/"},{"@type":"ListItem","position":5,"name":"Growth of Prototrophic E. coli Strains in D2O Minimal Salts Media"}]},{"@type":"WebSite","@id":"https:\/\/lsom.uthscsa.edu\/biochemistry\/#website","url":"https:\/\/lsom.uthscsa.edu\/biochemistry\/","name":"Biochemistry","description":"Department of Biochemistry and Structural Biology","potentialAction":[{"@type":"SearchAction","target":{"@type":"EntryPoint","urlTemplate":"https:\/\/lsom.uthscsa.edu\/biochemistry\/?s={search_term_string}"},"query-input":{"@type":"PropertyValueSpecification","valueRequired":true,"valueName":"search_term_string"}}],"inLanguage":"en-US"}]}},"_links":{"self":[{"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/pages\/1334","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/users\/161"}],"replies":[{"embeddable":true,"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/comments?post=1334"}],"version-history":[{"count":0,"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/pages\/1334\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/pages\/1008"}],"wp:attachment":[{"href":"https:\/\/lsom.uthscsa.edu\/biochemistry\/wp-json\/wp\/v2\/media?parent=1334"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}