IGEM:Harvard/2006/Cyanobacteria/Protocols

Media

1L agar for freshwater cyanobacteria (no glucose)

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

1. Mix 10 g agar and 1 mM thiosulfate (= 0.248 g), top off with H20 to 500 mL total volume.
2. Autoclave the product of (1)
3. Mix 20mL 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 plating PCC6803 cyanobacteria.

1. Mix 10 g agar and 1mM thiosulfate (= 0.248 g), top off with H20 to 500 mL total volume
2. Autoclave the product of (1)
3. Mix 20 mL 50x 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

1L liquid media for freshwater cyanobacteria

This protocol produces 1L of liquid media suitable for culturing PCC7942 and PCC6803.

1. Add 20 mL 50x BG-11 and 1 mM thiosulfate (= 0.248 g), top off with H20 to 1L total volume
2. Filter the solution into the desired container with a 0.2 µm filter.

Notes on growing freshwater cyanobacteria in liquid media

• Spray your gloves, containers, and working surfaces with ethanol, and let the ethanol dry, before handling cyanobacteria. We take this precaution because our bacteria do not have any antibiotic resistance as far as we know, so contamination is a bigger risk than with E. coli.
• The algae guide recommends growing bacteria in 125mL of media in a 250 mL Erlenmeyer flask (to give an idea of how high the flask should be filled).
• Use notched caps on the flasks, to permit gas flow while avoiding aerial contamination.
• When inoculating a culture, use autoclaved toothpicks to scrape the colony/streaks of interest, and throw them in the solution.
• According to Jeffrey Chabot's PhD thesis, cyanobacteria should be grown at 4200lux, cool white fluorescent lighting; this illumination can be higher during the initial growth stage.
• Do not completely cover the bacteria when growing them in the incubator. They need some airflow to exchange gas. Let the plates sit.

Freezing and thawing freshwater cyanobacteria

Freezing

1. Run the bacteria through a centrifuge to form a pellet
2. Place the pellet in a 7% DMSO solution, in a cryo-friendly tube
3. Freeze (-70C or -80C is fine)

Thawing

1. After taking the bacteria out of the freezer, pass them briefly through a stream of hot water or rub them with your fingers. This melts the solution around the walls of the tube.
2. Open the tube and jab the center of the frozen mass with a sterile toothpick, then pull the mass out, like pulling a popsicle out of a popsicle mold.
3. Immediately place the frozen bacteria in liquid media. Give a week or so for the bacteria to recover and regrow.

References

[1]: The use of thiosufate 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 |
3. Ohkawa H, Price GD, Badger MR, and Ogawa T. Mutation of ndh genes leads to inhibition of CO(2) uptake rather than HCO(3)(-) uptake in Synechocystis sp. strain PCC 6803. J Bacteriol. 2000 May;182(9):2591-6. DOI:10.1128/JB.182.9.2591-2596.2000 |

[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 |