An exogenous chloroplast genome for complex sequence manipulation in algae

Bryan M. O'Neill, Kari L. Mikkelson, Noel M. Gutierrez, Jennifer L. Cunningham, Kari L. Wolff, Shawn J. Szyjka, Christopher B. Yohn, Kevin Redding, Michael J. Mendez

    Research output: Contribution to journalArticle

    28 Citations (Scopus)

    Abstract

    We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.

    Original languageEnglish (US)
    Pages (from-to)2782-2792
    Number of pages11
    JournalNucleic Acids Research
    Volume40
    Issue number6
    DOIs
    StatePublished - Mar 2012

    Fingerprint

    Chloroplast Genome
    Genome
    Chlamydomonas reinhardtii
    Scenedesmus
    Chlorophyta
    Organism Cloning
    Yeasts
    Bacteria
    Genes

    ASJC Scopus subject areas

    • Genetics

    Cite this

    O'Neill, B. M., Mikkelson, K. L., Gutierrez, N. M., Cunningham, J. L., Wolff, K. L., Szyjka, S. J., ... Mendez, M. J. (2012). An exogenous chloroplast genome for complex sequence manipulation in algae. Nucleic Acids Research, 40(6), 2782-2792. https://doi.org/10.1093/nar/gkr1008

    An exogenous chloroplast genome for complex sequence manipulation in algae. / O'Neill, Bryan M.; Mikkelson, Kari L.; Gutierrez, Noel M.; Cunningham, Jennifer L.; Wolff, Kari L.; Szyjka, Shawn J.; Yohn, Christopher B.; Redding, Kevin; Mendez, Michael J.

    In: Nucleic Acids Research, Vol. 40, No. 6, 03.2012, p. 2782-2792.

    Research output: Contribution to journalArticle

    O'Neill, BM, Mikkelson, KL, Gutierrez, NM, Cunningham, JL, Wolff, KL, Szyjka, SJ, Yohn, CB, Redding, K & Mendez, MJ 2012, 'An exogenous chloroplast genome for complex sequence manipulation in algae' Nucleic Acids Research, vol. 40, no. 6, pp. 2782-2792. https://doi.org/10.1093/nar/gkr1008
    O'Neill BM, Mikkelson KL, Gutierrez NM, Cunningham JL, Wolff KL, Szyjka SJ et al. An exogenous chloroplast genome for complex sequence manipulation in algae. Nucleic Acids Research. 2012 Mar;40(6):2782-2792. https://doi.org/10.1093/nar/gkr1008
    O'Neill, Bryan M. ; Mikkelson, Kari L. ; Gutierrez, Noel M. ; Cunningham, Jennifer L. ; Wolff, Kari L. ; Szyjka, Shawn J. ; Yohn, Christopher B. ; Redding, Kevin ; Mendez, Michael J. / An exogenous chloroplast genome for complex sequence manipulation in algae. In: Nucleic Acids Research. 2012 ; Vol. 40, No. 6. pp. 2782-2792.
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