IGEM:MIT/2006/Notebook/2007-8-13

=Things to Do=

1. Was going to do another time course of J45120 and J45181 but could only find 13 flasks. Decided to do growth curves for J45200 and J45250 with isoamyl alcohol added initially (started 3:13 AM)

2. Set up J45200 250-mL culture to supplement with methyl salicylate later to develop extraction efficiencies (started 3:15 AM)- DONE

3. Put together package of "smelly strains" with J45120, J45200, isoamyl alcohol, and salicylic acid with instructions- WAS TOLD TO IGNORE REQUEST


 * Make stabs of J45200 and J45250 LCs


 * Make up extra salicylic acid solution


 * Write instructions


 * Make aliquots of isoamyl alcohol and salicylic acid

4. Miniprep and submit sequencing of J45250 1AT3 before 10 AM- DONE

5. Reply to Lily about GC- DONE (will be ready Sunday)

6. Ask Alex about DB-17 column/protocol used- DONE (supposed to meet with him at 1 PM tomorrow)

7. Clean up lab area- DONE

8. Look up safety info on isoamyl acetate/methyl salicylate/isoamyl alcohol/salicylic acid- DONE

9. Ask Drew 2 safety questions- DONE

10. GC extract J45200 cultures after adding varying amounts of methyl salicylate- DONE

Protocol:

Methyl Salicylate Stock Solution (made separate for each 20 mL of culture) contains 1 mL H20 and .05 g/100 mL (solubility limit=.07 g/100 mL)/1.174 g/mL/100 mL= .429 uL. Stock solution always vortexed

Add 20 mL of culture + X uL stock, where 1 ppm: X = 50, 2 ppm: X = 100, 5 ppm: X = 250, 10 ppm: X = 500, 15 ppm: X= 750, 20 ppm, X = 1000

11. Add .429 uL methyl salicylate to 25 mL of culture in 250-mL flask to give 16 ppm. Then let it shake at 110 RPM for 2 hours and GC extract (will later use this to determine decay rate of methyl salicylate)- DONE

12. Organize heptane waste bottles and fill in dates filled- DONE

13. Make new LCs of J45120, J45181, J45200, and J45250- DONE

14. Check sequencing from last week- DONE (only J45250 3K3 sequencing worked and it confirmed that the ligation went as planned)

15. Sign up for plate reader- DONE (Wednesday night)

16. Make graphs of growth curves for J45200 and J45250- DONE

=OD600s=

J45200

6 hrs- 0.18

7 hrs- 0.46

8 hrs- 0.84

9 hrs- 1.30

10 hrs- 1.64

11 hrs- 1.62

12 hrs- 1.62

13 hrs- 1.58

14 hrs- 1.58

J45250

6 hrs- 0.30

7 hrs- 0.68

8 hrs- 1.22

9 hrs- 1.52

10 hrs- 1.66

11 hrs- 1.79

12 hrs- 1.86

13 hrs- 1.84

14 hrs- 1.86