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Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris

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Abstract

Chlorella is a green unicellular alga that has a wide range of future biotechnical applications such as production of pharmaceuticals and biodiesel. Efficient genetic transformation of Chlorella vulgaris has been difficult due to technical limitations. In this study, an efficient and reliable transformation system of electroporation was established using two different reporter genes. First, C. vulgaris cell wall was digested with enzyme mixture for preparing protoplasts. The optimal transformation efficiency was 1.67 × 104 ± 0.083 cfu μg−1 plasmid under the following conditions: 2 × 106 cells mL−1 of growing culture; 655 V pulse voltage with 3.4 ms pulse width. After transformation, green and cyan fluorescence were observed from transgenic C. vulgaris harboring gfp (green fluorescent protein) gene of pCAMBIA1302 and cfp (cyan fluorescent protein) gene of pSK397, respectively, using laser confocal microscope. RT-PCR analysis as well as Southern blot confirmed the integration of reporter gene at the molecular level. This efficient transformation system of C. vulgaris would be valuable for the production of recombinant proteins in the future.

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Acknowledgments

This research was supported by grants from the LINK program, Korean Research Foundation, and from PAP program (PE17900) of Korea Polar Research Institute, Republic of Korea.

Authors’ contributions.

SRK conceived and designed study. MK conducted overall experiments, drafted manuscript and SRK finalized the manuscript. JJ maintained and measured the growth curves and analyzed transformants. JC conducted transformants analysis at protein level. All authors read and approved the final manuscript.

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  1. Department of Life Science, Sogang University, Seoul, 121-742, South Korea

    Manu Kumar, Jihyeong Jeon, Juyoung Choi & Seong-Ryong Kim

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  1. Manu Kumar
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Correspondence to Seong-Ryong Kim.

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Fig. S1

Vector map showing the cloning of CrCFP expression vector pSK397. (GIF 116 kb)

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(TIFF 474 kb)

Fig. S2

Expression of hygromycin phosphotransferase gene was analyzed in pCAMBIA1302 transformant of C. vulgaris. (GIF 51 kb)

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(TIFF 250 kb)

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Kumar, M., Jeon, J., Choi, J. et al. Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris. J Appl Phycol 30, 1735–1745 (2018). https://doi.org/10.1007/s10811-018-1396-3

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