Diatoms are unicellular photoautotrophic eukaryotes that play an important role in ecology by fixing large amounts of CO2 in the oceans. Because they evolved by secondary endocytobiosis—a process of uptake of a eukaryotic alga into another eukaryotic cell—they have a rather unusual cell biology and genetic constitution. Because the preparation of organelles is rather difficult as a result of the cytosolic structures, genetic transformation and expression of preproteins fused to green fluorescent protein (GFP) became one of the major tools to analyze subcellular localization of proteins in diatoms. Meanwhile several groups successfully attempted to develop genetic transformation protocols for diatoms. These methods are based on ‘‘biolistic’’ DNA delivery via a particle gun and allow the introduction and expression of foreign genes in the algae. Here a protocol for the genetic transformation of the diatom Phaeodactylum tricornutum is described as well as the subsequent characterization of the transformants.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Lee, R. E. (1989) Phycology, 2nd ed., Cambridge University Press, Cambridge, UK.
Norton, T. A., Melkonian, M., and Andersen, R. A. (1996) Algal biodiversity. Phycologia 35, 308–326.
Drum, R. W. and Gordon, R. (2003) Star Trek replicators and diatom nanotechnology. Trends Biotechnol. 21, 325–328.
Falkowski, P. G., Katz, M. E., Knoll, A. H., et al. (2004) The evolution of modern eukaryotic phytoplankton. Science 305, 354–360.
Delwiche, C. F. and Palmer, J. D. (1997) The origin of plastids and their spread via secondary symbiosis. Plant Syst. Evol. 11, 53–86.
Armbrust, E. V., Berges, J. A., Bowler, C., et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306, 79–86.
Maheswari, U., Montsant, A.., Goll, J., et al. (2005) The Diatom EST Database. Nucleic Acids Res. 33, D344–D347.
Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C., and Bowler, C. (1999) Transformation of nonselectable reporter genes in marine diatoms. Marine Biotechnology 1, 239-251.
Zaslavskaia, L. A., Lippmeier, J. C., Kroth, P. G., Grossman, A. R., and Apt, K. E. (2000) Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J. Phycol. 36, 379–386.
Poulsen, N. and Kröger, N. (2005) A new molecular tool for transgenic diatoms: control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette. FEBS J. 272, 3413–3423.
Dunahay, T. G., Jarvis, E. E., and Roessler, P. G. (1995) Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J. Phycol. 31, 1004–1012.
Poulsen, N., Chesley, P. M., and Kröger, N. (2006) Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). J. Phycol. 42, 1059–1065.
Kilian, O. and Kroth, P. G. (2005) Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids. Plant J. 41, 175–183.
Tanaka, Y., Nakatsuma, D., Harada, H., Ishida, M., and Matsuda, Y. (2005) Localization of soluble \beta -carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae. Plant Physiol. 138, 207-217.
Guillard, R. R. L. (1975) Culture of phytoplankton for feeding marine planktonic diatoms, in Culture of Marine Invertebrate Animals ( Smith, W. L. and Chanley, M. H., eds.), Plenum Press, New York, pp. 26–60.
Poulsen, N. and Kröger, N. (2005) A new molecular tool for transgenic diatoms: control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette. FEBS J. 272, 3413–3423.
Scala, S., Carels, N., Falciatore, A., Chiusano, M. L., and Bowler, C. (2002) Genome properties of the diatom Phaeodactylum tricornutum. Plant Physiol. 129, 993–1002.
Montsant, A., Jabbari, K., Maheswari, U., and Bowler, C. (2005) Comparative genomics of the pennate diatom Phaeodactylum tricornutum. Plant Physiol. 137, 500–513.
Acknowledgements
The author would like to thank Ansgar Gruber and Doris Ballert for careful reading of the manuscript and helpful suggestions. This work was supported by the German Research Foundation (DFG, project Kr 1661/3-1), the European Community (MARGENES, contract QLRT-2001-01226), and the University of Konstanz.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Kroth, P.G. (2007). Genetic Transformation. In: van der Giezen, M. (eds) Protein Targeting Protocols. Methods in Molecular Biology™, vol 390. Humana Press. https://doi.org/10.1007/978-1-59745-466-7_17
Download citation
DOI: https://doi.org/10.1007/978-1-59745-466-7_17
Publisher Name: Humana Press
Print ISBN: 978-1-58829-702-0
Online ISBN: 978-1-59745-466-7
eBook Packages: Springer Protocols