IGEM:MIT/2006/Notebook/2007-8-24

Redo of J45120/J45181 3-Hour Pulse

1. Add the following in a 2-L flask:

• 275 mL LB AMP/TET

• 27.5 uL grown J45120 culture

2. Grow the culture up for 20 mins at 220 RPM at 37C

3. Add the following in a 2-L baffled flask:

• 275 mL LB AMP/TET

• 27.5 uL grown J45181 culture

4. Grow the culture up for 20 mins at 220 RPM at 37C

5. Lower the RPM for both cultures to 110 RPM at 37C. The start of this step is Time Zero.

6. After x hours (for x=4 to x=12), extract 25 mL culture from master culture, pour into 125-mL flask, and add 0.5 mL .1 M salicylic acid to it for both J45120 and J45181 (grow up at 110 RPM)

7. At hour x+3 for hour x, GC extract culture

OD600s

Before Salicylic Acid Addition

J45120

Hour 4- 0.06

Hour 5-

Hour 6-

Hour 7-

Hour 8-

Hour 9-

Hour 10-

Hour 11-

Hour 12-

J45181

Hour 4-

Hour 5-

Hour 6-

Hour 7-

Hour 8-

Hour 9-

Hour 10-

Hour 11-

Hour 12-

After Salicylic Acid Addition

J45120

Hour 7-

Hour 8-

Hour 9-

Hour 10-

Hour 11-

Hour 12-

Hour 13-

Hour 14-

Hour 15-

J45181

Hour 7-

Hour 8-

Hour 9-

Hour 10-

Hour 11-

Hour 12-

Hour 13-

Hour 14-

Hour 15-

GC Results for Cumulative Methyl Salicylate

J45120

Hour 15- 175.9 ppm

Hour 16- 151.5 ppm

Hour 17- 129.6 ppm

J45181

Hour 15- 226.3 ppm

Hour 16- 123.5 ppm

Hour 17- 189.3 ppm

Weird, huh?

For the time being, will try repeating 3-hour pulse experiment