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The unicellular red alga Cyanidioschyzon merolae has been used as a eukaryotic photosynthetic model for various basic and applied studies. Although the nuclear genome of C. merolae can be modified by homologous recombination with exogenously introduced DNA, it has been difficult to modify multiple chromosome loci within the same strain because of the limited number of available positive selection markers. Recently, we reported a modified URA5.3 gene cassette (URA5.3T), which can be used repeatedly for nuclear genome transformation using the pMKT plasmid vectors for epitope tagging (3x FLAG- or 3x Myc-) of nuclear-encoded proteins. In addition, these plasmid vectors can also be used to knock out multiple genes one by one. This report describes the construction of DNA fragments for transformation and the detailed transformation procedure.

The unicellular red alga Cyanidioschyzon merolae has been used as a model photosynthetic eukaryote for various basic and applied studies, and several of these molecular genetics techniques have been reported. However, there are still improvements to be made concerning the plating method. The conventional plating method often generates diffuse colonies and single colonies cannot be easily isolated. To overcome these problems, we established a novel plating method for C. merolae, making use of melted cornstarch as the use of top agar plating in bacterial genetics. This method improved the formation of defined colonies in at least 4-fold higher efficiency than the conventional method, and made the handling procedure much easier than the previous method.