IGEM:MIT/2006/Notebook/2007-7-31

Set up for exponential phase extraction

• 25 uL overnight culture of J45181 in 250 mL LB A/T media (2-L flask)

• 25 uL overnight culture of J45120 in 250 mL LB A/T media (2-L flask)

• 2.5 uL overnight culture of J45800 in 25 mL LB A/K media (125-mL flask)

• 5 uL exponential-phase culture of B0015 in 25 mL LB A/K media (125-mL flask)

Grown up for 20 mins at 220 RPM, then switched to 110 RPM

J45120 6 hrs 0D600- 0.67, extract 6 mL, put at 4C for 2 hrs

J45181 6 hrs OD600- 0.45, extract 6 mL, put at 4C for 2 hrs

J45120 7 hrs OD600- 1.29, extract 6 mL, put at 4C for 1 hr

J45181 7 hrs OD600- 0.92, extract 6 mL, put at 4C for 1 hr

J45120 8 hrs OD600- 1.83, extract 6 mL

J45181 8 hrs OD600- 1.56, extract 6 mL

B0015 8 hrs OD600- 1.52, extract 6 mL

Other things to do

1. Phone meeting with Drew- DONE

2. Contact Alex about isoamyl acetate GC- DONE (awaiting reply)

3. Contact Dr. Tribe about media which will help overproduce chorismate in cells- DONE (awaiting reply)

4. Rework abstract for Amgen people- DONE

5. Rework Drew's edited abstract

6. Grow wild-type cells (MG1655) on M9 minimal media and see if they grow- DONE

• 24.25 mL minimal, .25 mL 40% gly, .5 mL casamino acids

• 24.25 mL minimal, .25 mL 40% glu, .5 mL casamino acids

• 23.5 mL minimal, 1 mL 20% fru, .5 mL casamino acids

• 8 mL minimal, .08 mL 40% gly

• 8 mL minimal, .08 mL 40% glu

• 8 mL minimal, .32 mL 20% fru

7. Grow J45700, J45800, and wild-type cells on Neidhardt media

8. Do protein gel sample preparation for J45120 (6 hr growth, 7 hr growth, 8 hr growth), J45181 (6 hr growth, 7 hr growth, 8 hr growth), & B0015- WORKING ON IT

9. At the end of the day, make LCs of J45995, J45996, B0015, R0040.E0840 to grow up for 16 hrs- DONE (at 6 PM)