IGEM:MIT/2006/Notebook/2007-6-28

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General Update

1. Talked with Professor Tribe of the University of Melbourne yesterday who was extraordinarily helpful. I am still in communication with him about how to best overproduce chorismate. For now, I will ask whether Yale's center has two types of strains suggested.

2. HPLC- Let's hope this works

3. Time courses looked decent, will modify the following:

  • 1. Will dilute a 5-mL grown culture for time courses from now on. Realized that before I took directly from the glycerol, which could have given rise to variations in growth between J45120 and J45181.
  • 2. Will take OD600 readings after GC sample extraction to reduce evaporation
  • 3. Will now move to two-hour time intervals to gain more data points.
  • 4. Will only go up to the 13-hour mark since cells seem to die after that or at least stopped producing significant amounts of methyl salicylate in both J45120 and J45181 cultures.
  • 5. So, the procedure would go as follows:

0:00- J45120 time course starts, add salicylic acid to a culture 1:00- extract that culture, 2:00- add salycilic acid to a culture, 3:00- extract that culture, 4:00- add salicylic acid to a culture, 5:00- extract that culture, 6:00- add salicylic acid to a culture, 7:00- extract that culture, 8:00- add salicylic acid to a culture, 9:00- extract that culture, 10:00- add salicylic acid to a culture, 11:00- extract that culture, *if necessary* 12:00- add salicylic acid to a culture, *if necessary* 13:00- extract that culture

0:20- J45181 time course starts, add salicylic acid to a culture 1:20- extract that culture, 2:20- add salycilic acid to a culture, 3:20- extract that culture, 4:20- add salicylic acid to a culture, 5:20- extract that culture, 6:20- add salicylic acid to a culture, 7:20- extract that culture, 8:20- add salicylic acid to a culture, 9:20- extract that culture, 10:20- add salicylic acid to a culture, 11:20- extract that culture, *if necessary* 12:20- add salicylic acid to a culture, *if necessary* 13:20- extract that culture

Things to Do Today

1. Add 15 uL grown 5-mL J45181 culture from last night to 150-mL LB AMP/TET solution (250-mL flasks) twice. Grow up for 20 mins at 220 RPM- DONE

2. Split the two cultures into 12 25-mL cultures in 12 250-mL flasks and grow up at 110 RPM- DONE

3. For every hour excluding the first hour for 12 hours (hours 2-13) take OD readings to construct growth curve as Francois suggested

4. Make new solution of water and isoamyl acetate (8.8 uL added per 20 mL water)- DONE

5. Run new solution on HPLC- RUNNING

6. Write back Professor Tribe to run by plan and confirm I heard him right on the phone- DONE

7. Contact Yale center about strains Tribe suggested

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