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.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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.
Harris EH (2008) The Chlamydomonas sourcebook, volume 1: introduction to Chlamydomonas and its laboratory use. Academic, San Diego, CAGoogle Scholar
Boynton JE, Gillham NW, Harris EH et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538PubMedCrossRefGoogle Scholar
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
Purton S (2007) Tools and techniques for chloroplast transformation of Chlamydomonas. Adv Exp Med Biol 616:34–45PubMedCrossRefGoogle Scholar
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
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
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
Werner R, Mergenhagen D (1998) Mating type determination of Chlamydomonas reinhardtii by PCR. Plant Mol Biol Rep 16:295–299CrossRefGoogle Scholar