Multiple Modification of Chromosomal Loci Using URA5.3 Selection Marker in the Unicellular Red Alga Cyanidioschyzon merolae

Tokiaki Takemura; Sousuke Imamura; Yuki Kobayashi; Kan Tanaka

doi:10.21769/BioProtoc.3204

Multiple Modification of Chromosomal Loci Using URA5.3 Selection Marker in the Unicellular Red Alga Cyanidioschyzon merolae

Bio Protoc. 2019 Apr 5;9(7):e3204. doi: 10.21769/BioProtoc.3204.

Authors

Tokiaki Takemura^{1

2}, Sousuke Imamura¹, Yuki Kobayashi¹, Kan Tanaka¹

Affiliations

¹ Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta 4259-R1-29, Yokohama 226-8503, Japan.
² School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta 4259-R1-29, Yokohama 226-8503, Japan.

Abstract

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.

Keywords: Cyanidioschyzon merolae; Epitope tagging; Gene knocking out; Homologous recombination; Selectable marker recycling; URA5.3.