IGEM:MIT/2006/Notebook/2007-7-3

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Things to Do

1. Request Japanese plasmid which overexpresses leucine in E. coli- DONE

2. Ask Professor Prather if she thinks that leucine is limiting in our reaction- DONE

3. Perform experiment that Reshma suggested (add .02 g/L (25 uL of 20 g/L stock) of leucine to J45600 and J45900 cultures (25 mL))- DONE

4. Resend request to Yale for aroF- aroG- aroH-, make sure it gets ordered today- DONE (AB3257 (aroF- aroG- aroH-) is on its way in today's mail!!!)

5. Ask Endy if they think that it is a good idea to add new promoters/RBSs to precursor devices in parallel with obtaining overexpressing strains/what overall goals should be a priority- DONE

6. Run GC samples from yesterday's time course- DONE (see below)

7. Meet with Alex about GC (2 PM)- DONE (need to prepare isoamyl acetate in heptane and heptane extraction of LB media, Alex seemed optimistic)

8. Print out GC procedure for isoamyl acetate for meeting- DONE

Results from Time Courses

J45120 (Constitutive Promoter)

Time (Hrs)------------OD600-----------Methyl Salicylate (ppm)---Methyl Salicylate (ppm)/OD600

3---------------------0.01------------NOT DETECTED-------------------------------------------

5---------------------0.21------------(-0.4)-----------------------------------------------------------

7---------------------1.09------------2.6------------------------------------------2.39

9--------------------2.5--------------16.9-----------------------------------------6.76

11--------------------2.5-------------12.8-----------------------------------------5.12

13--------------------2.8-------------11.8-----------------------------------------4.21

J45181 (Exponential-Phase Sensitive Promoter)

Time (Hrs)------------OD600-----------Methyl Salicylate (ppm)---Methyl Salyclate (ppm)/OD600

3---------------------0.01------------NOT DETECTED------------------------------------------

5---------------------0.27------------(0.1)------------------------------------------------------------

7---------------------1.15------------6.5-----------------------------------------5.65

9--------------------2.0--------------14.0----------------------------------------7.00

11--------------------3.0-------------11.5----------------------------------------3.83

13--------------------3.2-------------4.0-----------------------------------------1.25

Thoughts for next time:

Perhaps add in twice as many cells, so hour 5 is meaningful and hour 9 will likely be the beginning of stationary phase.

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