{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T13:22:43Z","timestamp":1787836963654,"version":"build-2784847793"},"reference-count":11,"publisher":"American Society for Microbiology","issue":"2","license":[{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Microbiol Biol Educ."],"published-print":{"date-parts":[[2026,8,27]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Molecular cloning is a fundamental technique in life science research. However, learning to perform traditional restriction-ligation-based methods is time-consuming and prone to failure, discouraging new students. Here, we describe a compact, two- to three-session undergraduate practical class using\n                      <jats:italic toggle=\"yes\">In vivo<\/jats:italic>\n                      Assembly, a simple, homology-based method that takes advantage of the native recombination machinery of\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      . Students swap a green fluorescent protein coding sequence with a red fluorescent protein coding sequence in a bacterial expression vector using PCR-amplified DNA fragments with short homology arms, followed by DpnI restriction enzyme digestion to remove template DNA, and then bacterial transformation. Success is directly visible by red versus green fluorescent bacterial colonies, providing rapid feedback and high student engagement. This low-cost protocol introduces key cloning concepts (PCR, homology design, template removal, bacterial transformation, and recombination) with reduced hands-on time and fewer failure points than classic approaches, making it ideal for effective teaching in resource-limited settings.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jmbe.00089-26","type":"journal-article","created":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T13:00:31Z","timestamp":1784120431000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["<i>In vivo<\/i>\n                    assembly to switch fluorescent protein expression in\n                    <i>Escherichia coli<\/i>\n                    : a simple laboratory class to introduce molecular cloning"],"prefix":"10.1128","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7384-2500","authenticated-orcid":false,"given":"Christopher J.","family":"Roberts","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01ee9ar58","id-type":"ROR","asserted-by":"publisher"}],"name":"Biodiscovery Institute, School of Medicine, University of Nottingham","place":["Nottingham, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7349-5513","authenticated-orcid":true,"given":"Kevin","family":"Gaston","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01ee9ar58","id-type":"ROR","asserted-by":"publisher"}],"name":"Biodiscovery Institute, School of Medicine, University of Nottingham","place":["Nottingham, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6500-9095","authenticated-orcid":false,"given":"Padma-Sheela","family":"Jayaraman","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01ee9ar58","id-type":"ROR","asserted-by":"publisher"}],"name":"Biodiscovery Institute, School of Medicine, University of Nottingham","place":["Nottingham, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1017\/bjt.2020.5"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.2144\/000114324"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1177\/0956797619881133"},{"key":"e_1_3_2_5_2","unstructured":"Puzzleheaded_192. 2024. My cloning is failing repeatedly and making my life hell. Available from: https:\/\/www.reddit.com\/r\/labrats\/comments\/1f04lp1\/my_cloning_is_failing_repeatedly_and_making_my"},{"key":"e_1_3_2_6_2","unstructured":"Jeff_98. 2023. [Rant] Frustrated over failed cloning. Available from: https:\/\/www.reddit.com\/r\/labrats\/comments\/171caxd\/rant_frustrated_over_failed_cloning"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.3791\/67067"},{"key":"e_1_3_2_8_2","first-page":"62","article-title":"A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction","volume":"10","author":"Jones DH","year":"1991","unstructured":"Jones DH, Howard BH. 1991. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. Biotechniques 10:62\u201366.","journal-title":"Biotechniques"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.REV119.009109"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.14655"},{"key":"e_1_3_2_11_2","unstructured":"van der Haar TP. 2019. Preparation and transformation of competent E. coli cells (CCMB80 method). Available from: https:\/\/www.protocols.io\/view\/preparation-and-transformation-of-competent-e-coli-5jyl8d38g2wp\/v1"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/jmbe.v20i3.1975"}],"container-title":["Journal of Microbiology &amp; Biology Education"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/jmbe.00089-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/jmbe.00089-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T13:03:19Z","timestamp":1787835799000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/jmbe.00089-26"}},"subtitle":[],"editor":[{"given":"Thiru","family":"Vanniasinkam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,27]]},"references-count":11,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2026,8,27]]}},"alternative-id":["10.1128\/jmbe.00089-26"],"URL":"https:\/\/doi.org\/10.1128\/jmbe.00089-26","relation":{},"ISSN":["1935-7877","1935-7885"],"issn-type":[{"value":"1935-7877","type":"print"},{"value":"1935-7885","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,27]]},"assertion":[{"value":"2026-04-02","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-06-19","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-15","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00089-26"}}