A Simple, Low-Cost Method for Chloroplast Transformation of the Green Alga Chlamydomonas reinhardtii

  • Chloe Economou
  • Thanyanan Wannathong
  • Joanna Szaub
  • Saul Purton
Part of the Methods in Molecular Biology book series (MIMB, volume 1132)


The availability of routine techniques for the genetic manipulation of the chloroplast genome of Chlamydomonas reinhardtii has allowed a plethora of reverse-genetic studies of chloroplast biology using this alga as a model organism. These studies range from fundamental investigations of chloroplast gene function and regulation to sophisticated metabolic engineering programs and to the development of the algal chloroplast as a platform for producing high-value recombinant proteins. The established method for delivering transforming DNA into the Chlamydomonas chloroplast involves microparticle bombardment, with the selection of transformant lines most commonly involving the use of antibiotic resistance markers. In this chapter we describe a simpler and cheaper delivery method in which cell/DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. Furthermore, we highlight the use of an expression vector (pASapI) that employs an endogenous gene as a selectable marker, thereby avoiding the contentious issue of antibiotic resistance determinants in transgenic lines.

Key words

Algae Chlamydomonas reinhardtii Chloroplast Glass beads Homologous recombination Transformation 



Research in the Purton lab into the genetic engineering of the Chlamydomonas chloroplast is funded by the UK’s Biotechnology and Biological Sciences Research Council and the “GIAVAP” and “SUNBIOPATH” FP7 projects of the European Union. SP acknowledges the equal contribution that CE and TW have made to this chapter.


  1. 1.
    Harris EH (2008) The Chlamydomonas sourcebook, volume 1: introduction to Chlamydomonas and its laboratory use. Academic, San Diego, CAGoogle Scholar
  2. 2.
    Boynton JE, Gillham NW, Harris EH et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538PubMedCrossRefGoogle Scholar
  3. 3.
    Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucl Acids Res 19:4083–4089PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Purton S (2007) Tools and techniques for chloroplast transformation of Chlamydomonas. Adv Exp Med Biol 616:34–45PubMedCrossRefGoogle Scholar
  5. 5.
    Michelet L, Lefebvre-Legendre L, Burr SE et al (2011) Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas. Plant Biotechnol J 9:565–574PubMedCrossRefGoogle Scholar
  6. 6.
    Surzycki R, Cournac L, Peltier G et al (2007) Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci U S A 104:17548–17553PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Specht E, Miyake-Stoner S, Mayfield S (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32:1373–1383PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Radakovits R, Jinkerson RE, Darzins A et al (2010) Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 9:486–501PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87:1228–1232PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Kindle KL, Richards KL, Stern DB (1991) Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88:1721–1725PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Werner R, Mergenhagen D (1998) Mating type determination of Chlamydomonas reinhardtii by PCR. Plant Mol Biol Rep 16:295–299CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Chloe Economou
    • 1
  • Thanyanan Wannathong
    • 2
  • Joanna Szaub
    • 1
  • Saul Purton
    • 1
  1. 1.Institute for Structural and Molecular BiologyUniversity College LondonLondonUK
  2. 2.Department of BiologySilpakorn UniversityNakornpathomThailand

Personalised recommendations