IGEM:MIT/2006/Notebook/2007-8-9

Time Course Protocol

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

• 250 mL LB AMP/TET

• 5 mL 0.1 M Salicylic Acid

• 25 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:

• 250 mL LB AMP/TET

• 5 mL 0.1 M Salicylic Acid

• 25 uL grown J45181 culture

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

5. Split the J45120 culture into 9 25-mL cultures in 125-mL flasks and grow at 110 RPM at 37C

6. Split the J45181 culture into 9 25-mL cultures in 125-mL flasks and grow at 110 RPM at 37C

7. For both J45120 and J45181 cultures, take out one culture every hour for hours 5-13 of growth and GC extract

Things to Do

1. Time Course

2. Grow up a culture of J45200 and J45250 with added precursor- DONE and smell during exponential and stationary phase

3. Make the ligations and transformations outlined in yesterday's notebook entry- DONE

4. Streak out J45100- DONE

OD600s for Time Course

J45120

5 hrs- 0.08

6 hrs- 0.22

7 hrs- 0.60

8 hrs- 0.94

9 hrs- 1.34

10 hrs- 1.50

11 hrs- 1.66

12 hrs- 1.72

13 hrs- 1.72

J45181

5 hrs- 0.08

6 hrs- 0.20

7 hrs- 0.58

8 hrs- 0.88

9 hrs- 1.14

10 hrs- 1.54

11 hrs- 1.68

12 hrs- 1.50

13 hrs- 1.70