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 Edward Redding, Michael J. Mendez

Research output: Contribution to journalArticle

29 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
Pages (from-to)2782-2792
Number of pages11
JournalNucleic Acids Research
Volume40
Issue number6
DOIs
Publication statusPublished - 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 Edward; 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, KE & 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 Edward ; 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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