title: A simple, low-cost method for chloroplast transformation of the green alga Chlamydomonas reinhardtii.
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.
authors.affiliation: Institute for Structural and Molecular Biology, University College London, London, UK.
authors.forename: Chloe
authors.initials: C
authors.lastname: Economou
authors.forename: Thanyanan
authors.initials: T
authors.lastname: Wannathong
authors.forename: Joanna
authors.initials: J
authors.lastname: Szaub
authors.forename: Saul
authors.initials: S
authors.lastname: Purton
mesh_terms: D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D005898:Glass; D045332:Photosystem II Protein Complex / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins