CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome.
View / Open Files
Publication Date
2022-05-04Journal Title
Biotechnology Journal
ISSN
1860-6768
Publisher
Wiley
Pages
e2200088
Type
Article
This Version
AM
Physical Medium
Print-Electronic
Metadata
Show full item recordCitation
Jackson, H. O., Taunt, H. N., Mordaka, P. M., Kumari, S., Smith, A., & Purton, S. (2022). CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome.. Biotechnology Journal, e2200088. https://doi.org/10.1002/biot.202200088
Abstract
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 markers'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. This article is protected by copyright. All rights reserved.
Keywords
5-fluorocytosine, Chlamydomonas, chloroplast, marker recycling, plastome
Sponsorship
BBSRC (via University College London (UCL)) (FCAGF)
Biotechnology and Biological Sciences Research Council (BB/R01860X/1)
Biotechnology and Biological Sciences Research Council (BB/L002957/1)
Biotechnology and Biological Sciences Research Council (BB/I00680X/1)
Biotechnology and Biological Sciences Research Council (BB/L014130/1)
Embargo Lift Date
2023-05-04
Identifiers
External DOI: https://doi.org/10.1002/biot.202200088
This record's URL: https://www.repository.cam.ac.uk/handle/1810/337118
Statistics
Total file downloads (since January 2020). For more information on metrics see the
IRUS guide.
Recommended or similar items
The current recommendation prototype on the Apollo Repository will be turned off on 03 February 2023. Although the pilot has been fruitful for both parties, the service provider IKVA is focusing on horizon scanning products and so the recommender service can no longer be supported. We recognise the importance of recommender services in supporting research discovery and are evaluating offerings from other service providers. If you would like to offer feedback on this decision please contact us on: support@repository.cam.ac.uk