IGEM:Harvard/2006/Cyanobacteria/Protocols

Media

1L agar for freshwater cyanobacteria (no glucose)

This protocol produces 1L agar suitable for freshwater cyanobacteria (PCC7942 and PCC6803). [1]

1. Mix 10 g of agar and 1 mL thiolsulfate, top off with H20 to 500 mL total volume.
2. Autoclave the product of (1)
3. Mix 20mL of 50x BG-11 solution and 480 mL of H20
4. Autoclave the product of (3)
5. Mix (2) and (4), pour plates and let cool

1L agar for freshwater cyanobacteria (with glucose)

This protocol produces 1L agar with 5 mM glucose suitable for PCC6803 cyanobacteria.

1. Mix 10 g of agar and 1mL thiolsulfate, top off with H20 to 500 mL total volume
2. Autoclave the product of (1)
3. Mix 20 mL of 50x Sigma BG-11 solution and 230 mL of H20
4. Autoclave the product of (3)
5. Mix 5 mL glucose and 245 mL of H20. [2]
6. Autoclave the product of (5)
7. Mix (2) and (4) and (6), pour plates and let cool

Growth in liquid culture (freshwater)

According to Chabot's PhD thesis, grow them at 4200lux; can be higher during initial growth stage. The algae guide said to grow in 125mL of media in an Earlemeyer flask. Be sure to spray down with ethanol beforehand. Used autoclaved toothpicks and threw in the solution. On shaker; do not cover. Let plates sit.

References

[1]: The use of thiosulfate is discussed in the following papers; it is isn't required, but it supposedly improves growth rate.

1. Thiel T, Bramble J, and Rogers S. Optimum conditions for growth of cyanobacteria on solid media. FEMS Microbiol Lett. 1989 Oct 1;52(1-2):27-31. DOI:10.1016/0378-1097(89)90164-x |

[2]: Usage of glucose is to take advantage of heterotrophic growth of PCC6803 with glucose; this supposedly cuts down the growth cycle to 4 days, according to the 2006 MIT iGEM team.

2. Spence E, Bailey S, Nenninger A, Møller SG, and Robinson C. A homolog of Albino3/OxaI is essential for thylakoid biogenesis in the cyanobacterium Synechocystis sp. PCC6803. J Biol Chem. 2004 Dec 31;279(53):55792-800. DOI:10.1074/jbc.M411041200 |