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- DONE (will give a run a try tomorrow)

4. Perform a time course in which I follow this protocol with J45181- WORKING ON IT:

  • 1. Grow a 150-mL culture up at 220 RPM for 20 mins at 37 degrees C
  • 2. Split culture into 6 25-mL cultures and grow up at 150 RPM (previously 110 RPM) at 37 degrees C
  • 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 (1:15 PM OD600=0.00 at 2:15 PM, 5:15 PM OD600=0.02 at 6:15 PM, 9:15 PM, 1:15 AM, 5:15 AM, 9:15 AM)

5. Grow up a culture of J45120 for at 220 RPM, add 2 mM SA, and see if it smells after 60 mins- could smell it a bit after only 35 min

6. Check sequencing of internal primers- DONE (600D1 looks good, 800D1 has an added "AAT" in the middle of it, 600D2 doesn't look good, for now... keep glycerols of 600D1 and 800D1)

7. Sequence J45181 with internal primers to see if J45800 has a new mutation ("AAT") that I should be worried about- DONE

8. Contact Pittard and Tribe about overproducing chorismate in E. coli- DONE (they're still active it seems; got a response from Tribe who said he'll talk with Pittard)

9. Grow up J45200 with isoamyl alcohol tonight plus culture without isoamyl alcohol to be spiked with isoamyl acetate tomorrow for HPLC- DONE

10. Find solubility of leucine and make up solution- DONE (link: http://www3.interscience.wiley.com/cgi-bin/fulltext/108066360/PDFSTART)

11. Grow up J45600 and J45900 cultures with L-Leucine added

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