Top Starch Plating Method for the Efficient Cultivation of Unicellular Red Alga Cyanidioschyzon merolae

Materials and Reagents

1. Pipette tips (NIPPON Genetics Co., Ltd., catalog numbers: FG-102, FG-301, FG-401)
2. Parafilm (LMS Co., Ltd., catalog number: PF, PM-996)
3. 1.5 ml microtube (BM Equipment Co., Ltd., catalog number: NT-175)
4. 2.0 ml microtube (WATSON Co., Ltd., catalog number: 132-620C)
5. 50 ml centrifuge tube (Corning Incorporated., catalog number: 352196)
6. AnaeroPouch (Mitsubishi Gas, catalog number: 2-3764-02)
7. AnaeroPack (Mitsubishi Gas, catalog number: 2-3765-01)
8. Sterilized plastic plate (Ø 90 mm) (As One Corporation., catalog number: GD90-15)
9. Cyanidioschyzon merolae 10D [available as NIES-3377 from NIES collection, Tsukuba, Japan (http://mcc.nies.go.jp/index_en.html)]
   
10. Cornstarch (Kawamitsu-Bussan, catalog number: 4901 486 02701 6)
11. Gellan gum (FUJIFILM Wako Pure Chemical Corporation, catalog number: 073-03071)
12. Uracil (Sigma-Aldrich, catalog number: U0750-5G)
13. 5-Fluoroorotic acid monohydrate (5-FOA) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 003234)
14. Ethanol (FUJIFILM Wako Pure Chemical Corporation, catalog number: 055-00457)
    
15. (NH₄)₂SO₄ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 019-03435)
16. MgSO₄∙7H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 138-00415)
17. H₂SO₄ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 192-04696)
18. H₃BO₃ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 021-02195)
19. MnCl₂∙4H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 133-00725)
20. ZnCl₂ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 268-01022)
21. Na₂MoO₄∙2H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 198-02471)
22. CoCl₂∙6H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 038-03681)
23. CuCl₂∙2H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 039-04135)
24. KH₂PO₄ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 169-04245)
25. CaCl₂∙2H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 038-19735)
26. FeCl₃∙6H₂O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 090-02802)
27. Na₂EDTA (FUJIFILM Wako Pure Chemical Corporation, catalog number: 345-01865)
28. 20% slurry cornstarch stock solution (see Recipes)
29. MA2 medium (Kobayashi et al., 2010) (see Recipes)
    
    1. MA2 solution I
    2. A6 minor salts
       
    3. MA2 solution II
    4. MA2 solution III
    5. MA2 solution IV
30. MA2 solid gellan gum plate (see Recipes)
    

Equipment

1. Pipettes (Gilson, Inc., catalog numbers: F123600, F123601, F123602)
2. Refrigerated centrifuge (Koki Holdings Co., Ltd., model: CF16RXII)
3. Microcentrifuge (TOMY Seiko Co., Ltd., model: MX150)
4. 500 ml flask (IWAI, catalog number: 4442FK500)
5. Heat block (Chiyoda Science Co., Ltd., model: MiniT-100)
6. Bio incubator (TOMY Seiko Co., Ltd., model: CLE-303)
7. Clean bench (Panasonic Healthcare Co., Ltd., model: MCV-131BNF)
8. Vortex mixer (M & S Instruments Inc., model: SI-0286)
9. Sterile filter (Ø 0.22 μm, Merck, catalog number: SLGV033RS)
   

Procedure

1. Prepare the MA2 solid gellan gum plate and the 20% slurry cornstarch stock solution (see Recipes).
2. Mix 100 μl of the cornstarch slurry stock solution and 900 μl of sterile dH₂O in a 2 ml microtube.
3. Centrifuge the microtubes at 500 x g for 1 min at room temperature and discard the supernatant.
4. Add 900 μl of sterile dH₂O and mix vigorously.
5. Centrifuge again at 500 x g for 1 min at room temperature and discard the supernatant.
6. Add 900 μl of sterile dH₂O and mix vigorously.
7. Incubate the microtube at 98 °C for 10 min but continue to mix thoroughly by inverting the tube every 2 min.
8. Cool down at room temperature for 10 min (= top starch solution).
   Note: Steps 8-11 should be performed in a laminar flow cabinet.
9. Add 1 ml of MA2 culture medium containing C. merolae cells (in case of wild type, 500 cells by microscopy observation) to the top starch solution and mix gently.
10. Pour the mixture onto the surface of the MA2 solid gellan gum plate and spread the mixture over the whole plate area by tilting the plate to spread the top starch solution uniformly.
11. Leave the plate open for 15-20 min to allow the top starch solution to solidify. After this step, the surface of the top starch is still wet, and the plate should be incubated with the top starch solution pointing upwards for the entire time.
12. Incubate the MA2 solid gellan gum plate at 40 °C under continuous white light (35-50 μmol m^-2 s^-1) supplemented with 5% CO₂ in AnaeroPack with AnaeroPouch.
13. After 2-4 weeks, colonies of the transformed cells appear on the MA2 solid gellan gum plate.
    

Recipes

1. 20% slurry cornstarch stock solution (Fujiwara and Ohnuma, 2018)
   
   1. Add 10 g of cornstarch and 40 ml of dH₂O to the 50 ml conical tube
   2. Mix the tube well using a vortex mixer
   3. Centrifuge the tube at 1,200 x g for 5 min at 4 °C
   4. Discard the supernatant by decantation
   5. Resuspend the pellet in 40 ml of 100% of ethanol using a vortex mixer
   6. Centrifuge the tube at 1,200 x g for 5 min at 4 °C
   7. Discard the supernatant by decantation
   8. Fill up to 50 ml with 75% of ethanol
   9. Store the tube at 4 °C, wrapped using parafilm until use
2. MA2 medium
   
   1. Mix 100 ml of MA2 solution I, 10 ml of MA2 solution II, 1 ml of MA2 solution III and 885 ml of dH₂O in a glass bottle and sterilize by autoclaving
   2. Add 4 ml of MA2 solution IV to the mixture in a laminar flow cabinet
   
   MA2 solution I
   
   
   A6 minor salts
   
   
   MA2 solution II
   
   
   MA2 solution III
   
   
   MA2 solution IV
   
   Note: This MA2 solution IV should be sterilized by filtration and stored at 4 °C in the dark.
   
   
3. MA2 solid gellan gum plate (for making 9-10 plates)
   
   1. Mix 30 ml of MA2 solution I, 3 ml of MA2 solution II, 300 μl of MA2 solution III, and 70 ml of dH₂O in a 500 ml flask. Mix 200 ml of dH₂O and 1.5 g of gellan gum in another 500 ml flask. Sterilize these flasks by autoclaving, and subsequent plate preparation should be performed in a laminar flow cabinet
   2. After autoclaving, mix both solutions and add 1.2 ml of MA2 solution IV. In case of 5-FOA selection to isolate the URA deficient mutants (for example applying URA marker recycling system, Takemura et al., 2018; Takemura et al., 2019), add directly 150 mg of uracil (final concentration: 0.5 mg/ml) and 240 mg of 5-FOA (final concentration: 0.8 mg/ml) in powder to the flask
   3. Pour the mixed solution into the sterilized plastic plates
   4. Dry the plates at room temperature for 15 min
   5. Store at 4 °C in the dark

Acknowledgments

This protocol was adapted from Takemura et al. (2018). The authors thank Y. Ide for technical assistance. This study was supported by MEXT/JSPS KAKENHI (Grant numbers: 17K07438 to S.I., 17K07439 to Y.K.) and by Advanced Low Carbon Technology Research and Development Program (ALCA) of Japan Science and Technology Agency (JST) to K.T.

Competing interests

No competing interests declared.

References

1. Fujiwara, T. and Ohnuma, M. (2018). Procedures for transformation and their applications in Cyanidioschyzon merolae. In: Kuroiwa, T., Miyagishima, S.Y., Matsunaga, S., Sato, N., Nozaki, H., Tanaka, K. and Misumi, O. (Eds.). Cyanidioschyzon merolae. Singapore, Springer Singapore: 87-103.
2. Imamura, S., Kanesaki, Y., Ohnuma, M., Inouye, T., Sekine, Y., Fujiwara, T., Kuroiwa, T. and Tanaka, K. (2009). R2R3-type MYB transcription factor, CmMYB1, is a central nitrogen assimilation regulator in Cyanidioschyzon merolae. Proc Natl Acad Sci U S A 106(30): 12548-12553.
3. Imamura, S., Terashita, M., Ohnuma, M., Maruyama, S., Minoda, A., Weber, A. P., Inouye, T., Sekine, Y., Fujita, Y., Omata, T. and Tanaka, K. (2010). Nitrate assimilatory genes and their transcriptional regulation in a unicellular red alga Cyanidioschyzon merolae: genetic evidence for nitrite reduction by a sulfite reductase-like enzyme. Plant Cell Physiol 51(5): 707-717.
4. Kobayashi, Y., Ohnuma, M., Kuroiwa, T., Tanaka, K., and Hanaoka, M. (2010). The basics of cultivation and molecular genetic analysis of the unicellular red alga Cyanidioschyzon merolae. Endocytobiosis Cell Res 20: 53-61.
5. Matsuzaki, M., Misumi, O., Shin-I, T., Maruyama, S., Takahara, M., Miyagishima, S. Y., Mori, T., Nishida, K., Yagisawa, F., Nishida, K., et al. (2004). Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428(6983): 653-657.
6. Minoda, A., Sakagami, R., Yagisawa, F., Kuroiwa, T. and Tanaka, K. (2004). Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 45(6): 667-671.
7. Ohnuma, M., Yokoyama, T., Inouye, T., Sekine, Y. and Tanaka, K. (2008). Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 49(1): 117-120.
8. Shimogawara, K., Fujiwara, S., Grossman, A. and Usuda, H. (1998). High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148(4): 1821-1828.
9. Takemura, T., Imamura, S., Kobayashi, Y. and Tanaka, K. (2018). Construction of a selectable marker recycling system and the use in epitope tagging of multiple nuclear genes in the unicellular red alga Cyanidioschyzon merolae. Plant Cell Physiol 59(11): 2308-2316.
10. Takemura, T., Imamura, S., Kobayashi, Y. and Tanaka, K. (2019). Preparation of introducing DNA fragment for multiple modification of chromosomal loci in the unicellular red alga Cyanidioschyzon merolae. Bio-protocol (In-review).
11. Zienkiewicz, M., Krupnik, T., Drożak, A., Golke, A. and Romanowska, E. (2017a). Chloramphenicol acetyltransferase-a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae. Protoplasma 254(1): 587-596.
12. Zienkiewicz, M., Krupnik, T., Drożak, A., Golke, A. and Romanowska, E. (2017b). Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments. Plant Mol Biol 93(1-2): 171-183.
13. Zienkiewicz, M., Krupnik, T., Drożak, A., Wasilewska, W., Golke, A. and Romanowska, E. (2018). Deletion of psbQ' gene in Cyanidioschyzon merolae reveals the function of extrinsic PsbQ' in PSII. Plant Mol Biol 96(1-2): 135-149.