{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T14:32:34Z","timestamp":1786977154327,"version":"build-2736575974"},"reference-count":49,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2022,5,12]],"date-time":"2022-05-12T00:00:00Z","timestamp":1652313600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotechnology Journal"],"published-print":{"date-parts":[[2022,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The chloroplast represents an attractive compartment for light\u2010driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( =\u00a0plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations. This presents issues with respect to selectable markers, since there are a limited number available, they can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative selection system, known as CpPosNeg, for the green microalga\n                    <jats:italic>Chlamydomonas reinhardtii<\/jats:italic>\n                    that allows creation of marker\u2010free transformants starting from wild\u2010type strains. The system employs a dual marker\u00a0encoding a fusion protein of\n                    <jats:italic>E. coli<\/jats:italic>\n                    aminoglycoside adenyltransferase (AadA: conferring spectinomycin resistance) and a variant of\n                    <jats:italic>E. coli<\/jats:italic>\n                    cytosine deaminase (CodA: conferring sensitivity to 5\u2010fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5\u2010fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the 3\u2032UTR of the marker and the 3\u2032UTR of the introduced transgene. We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.\n                  <\/jats:p>","DOI":"10.1002\/biot.202200088","type":"journal-article","created":{"date-parts":[[2022,5,5]],"date-time":"2022-05-05T00:08:43Z","timestamp":1651709323000},"source":"Crossref","is-referenced-by-count":17,"title":["CpPosNeg: A positive\u2010negative selection strategy allowing multiple cycles of marker\u2010free engineering of the Chlamydomonas plastome"],"prefix":"10.1002","volume":"17","author":[{"given":"Harry O.","family":"Jackson","sequence":"first","affiliation":[{"name":"Department of Structural and Molecular Biology University College London  London UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henry N.","family":"Taunt","sequence":"additional","affiliation":[{"name":"Department of Structural and Molecular Biology University College London  London UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pawe\u0142 M.","family":"Mordaka","sequence":"additional","affiliation":[{"name":"Department of Plant Sciences University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sujata","family":"Kumari","sequence":"additional","affiliation":[{"name":"DBT\u2010ICT Centre for Energy Biosciences Institute of Chemical Technology  Mumbai India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alison G","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Plant Sciences University of Cambridge  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9342-1773","authenticated-orcid":false,"given":"Saul","family":"Purton","sequence":"additional","affiliation":[{"name":"Department of Structural and Molecular Biology University College London  London UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,5,12]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"crossref","unstructured":"Jensen P. 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