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Chloroplast Biotechnology

Volume 1132 of the series Methods in Molecular Biology pp 401-411

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

  • Chloe EconomouAffiliated withInstitute for Structural and Molecular Biology, University College London
  • , Thanyanan WannathongAffiliated withDepartment of Biology, Silpakorn University
  • , Joanna SzaubAffiliated withInstitute for Structural and Molecular Biology, University College London
  • , Saul PurtonAffiliated withInstitute for Structural and Molecular Biology, University College London

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Abstract

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