IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-6-26

Incubator status

Our incubator now houses a total of 11 cultures and 2 plates. The cultures from 2006-6-22 are stored in 250 mL Erlenmeyer flasks with 125 mL of liquid medium, and airhole-punched parafilm covering the top.

Shaking floor (counter-clockwise from lower left)

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1. Control
2. PCC 7942 single colony from 2006-6-22
3. PCC 6803 plate
4. PCC 6803 single colony
5. PCC 7942 streak from 2006-6-22
6. PCC 6803 streak
7. PCC 6803 streak from 2006-6-22
8. PCC 7942 streak from the dried PCC 7942 plate
9. PCC 6803 single colony
10. PCC 7942 plate (dried)

Upper shelf (not directly lit, non-shaking, left to right)

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1. PCC 6803 streak
2. Control from 2006-6-22
3. PCC 6803 single colony from 2006-6-22

New cultures

We inoculated 5 cultures in 500 mL Erlenmeyer flasks with "breathable" caps, using 125 mL of the medium described here.

• 2 flasks had PCC 6803 streaks
• 1 flask had PCC 6803 single colony
• 1 flask had what we could scrape from our dried PCC 7942 plate
• 1 flask for control

Loss of PCC 7942 plate

Our PCC 7942 agar plate dried out between Thursday evening and Friday morning. There is no more agar on the plate, and the bacteria appear to have died. We will have to get more PCC 7942 from somewhere.

Growth conditions research

See IGEM:Harvard/2006/Conditions/Cyanobacteria

Thiosulfate stock

• We created a 500x stock of 1mM/L thiosulfate solution.
  • 1M thiosulfate = 248.2 g
  • 1M/L thiosulfate = 248.2 g/L
  • 1mM/L thiosulfate = 0.248 g/L
  • Our stock is 24.82 g in 200 mL thiosulfate = 500 mM/L

Fungus among us

Our flasks were contaminated by fungi over the weekend. We will need to reinoculate our cultures.

:(

Results

Single colony 6803

• Cloudy media
• Visible greenish-yellow material (floating around)

Streak-colony 6803

• White bulbous growth on one end of toothpick with a slight green center.
• Could be fungus competing with 6803
• Clear media

Streak-colony 7942

• Clear media
• Barely-visible material

Single colony 7942

• Clear media
• Barely-visible material

Single colony 6803

• No toothpick in the flask: a streaker was used instead
• Clear media
• Visible particles growing (slightly green)
• Big clear particles

Additional notes:

• All cultures were grown in B6-11 media
• Grown on 6/22/06
• All of the parafilm caps broke over the course of the weekend
• Grown in 30°C, under 4000 lux
• Shaker may have been off/very slow for 4 days; didn't want flasks to fall out of holders.

Summary

• Obviously, cyanobacteria growth was not optimal. Need to create new cultures but hopefully with different parameters.
• Will continue to grow 6803 single and streak colonies for several days, as these showed the most promise.
• Ideas for new cultures:
  • Turn off fan -> can't because incubator needs the fan to regulate the temperature
  • Use different flasks -> Probably will switch to 125mL or 500mL flasks with notched caps
    • Notched caps will allow us to keep caps on loosely and still get airflow into the flask. Might help us avoid contamination.
    • Also considering tissue-culture flasks, with air-filter on caps. Need to figure out how to get those to fit and stay secure in incubator.
  • Toothpicks -> May not have been sterile. Will use different set that were definitely autoclaved.
  • Lighting -> Peng is currently researching different papers to see if any different lighting was used. 4000 lux might be too much.
    • Light Frequency -> May be a problem but doesn't look like it after a literature review. Could be local variations, but that would be hard to pinpoint/change.
  • Temperature -> 30°C may be too warm; Peng has seen 20°-30° C in papers.