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CpPosNeg: a positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome
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  • Harry Jackson,
  • Henry Taunt,
  • Pawel Mordaka,
  • Sujata Kumari,
  • Alison Smith,
  • Saul Purton
Harry Jackson

Corresponding Author:h.o.jackson@ucl.ac.uk

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Henry Taunt
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Pawel Mordaka
Cambridge University
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Sujata Kumari
Institute of Chemical Technology
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Alison Smith
University of Cambridge
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Saul Purton
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The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome (=plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations, and this will present issues with respect to selectable markers: there are a limited number of markers available, these 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 marker system for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the marker’s 3’UTR and the 3’UTR of the introduced transgene(s). We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.
15 Feb 2022Submitted to Biotechnology Journal
17 Feb 2022Submission Checks Completed
17 Feb 2022Assigned to Editor
21 Mar 2022Reviewer(s) Assigned
11 Apr 2022Editorial Decision: Revise Minor
19 Apr 20221st Revision Received
20 Apr 2022Submission Checks Completed
20 Apr 2022Assigned to Editor
23 Apr 2022Editorial Decision: Accept
Oct 2022Published in Biotechnology Journal volume 17 issue 10 on pages 2200088. 10.1002/biot.202200088