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

=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

3-0.01NOT DETECTED---

5-0.21(-0.4)---

7-1.092.6--2.39

92.5--16.9-6.76

112.5-12.8-5.12

132.8-11.8-4.21

J45181 (Exponential-Phase Sensitive Promoter)

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

3-0.01NOT DETECTED--

5-0.27(0.1)

7-1.156.5-5.65

92.0--14.07.00

113.0-11.53.83

133.2-4.0-1.25

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.