IGEM:MIT/2006/Notebook/2007-6-25

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Status

1. Waiting to hear back from Prather and Frost about upregulating chorismate

2. Waiting to meet with Andreas from Sauer lab on LC

Things to Do Today

1. Contact Andreas asking about a meeting today- DONE

2. Perform a back-of-the-envelope calculation on how long it would take our enzyme to make substantial amounts of methyl salicylate for pulse-chase- DONE

Used kcat and Km values from 6/6/06 notebook page, Michaelis-Menten Equation, and assumed a thousand times less enzyme than substrate (as looked reasonable from http://web.indstate.edu/thcme/mwking/enzyme-kinetics.html) and decided that an hour is a good time to wait for salicylic acid to be converted to methyl salicylate (~40% efficiency)

I also remember us doing an experiment where the cultures smelled after <1 hr, although I couldn't find it in the notebook.

3. Have a meeting with Andreas

4. Perform a time course in which I follow this protocol with J45181:

  • 1. Grow a 150-mL culture up at 220 RPM for 30 mins.
  • 2. Split culture into 6 25-mL cultures and grow up at 150 RPM (previously 110 RPM)
  • 3. Add 2 mM salicylic acid immediately to 1 culture
  • 4. After 1 hr, remove the culture, take OD600 reading, and extract 20 mL of culture using procedure outlined last week
  • 5. Repeat every 4 hrs to 20 hrs (12:30 PM, 4:30 PM, 8:30 PM, 12:30 AM, 4:30 AM, 8:30 AM)

5. Grow up a culture of J45120 for 45 mins at 220 RPM, add 2 mM SA, and see if it smells after 45 min

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