IGEM:MIT/2006/Notebook/2007-8-28

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J45996/I7100 3-Hour Pulse

1. Add the following in 2 2-L flasks:

  • 275 mL LB + Antibiotic
  • 27.5 uL grown I7100 or J45996 culture

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

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

4. At Time Zero, add .5 mL .1 M salicylic acid to a 125-mL flask and add 25 mL culture. Remove 3 200 uL aliquots for each culture for every 3 hours for hour 6, 9, 12, and 15.

6. After x hours (for x=4 to x=12 where x is even), extract 25 mL culture from master culture, pour into 125-mL flask, and add 0.5 mL .1 M salicylic acid to it for all cultures (grow up at 110 RPM). Also remove 3 200 uL aliquots for each culture and run on the plate reader.

7. At hour x+3 for hour x, remove 3 200 uL aliquots of each culture from

What this means at each hour for each culture

Hour 4 (12:15)- remove 25 mL from 2-L flask and add salicylic acid, remove 3 200 uL aliquots from 2-L flask

Hour 6 (2:15)- remove 25 mL from 2-L flask and add salicylic acid, remove 3 200 uL aliquots from 2-L flask

Hour 7 (3:15)- remove 3 200 uL aliquots from 125-mL flask (salicylic acid added at Hour 4 and Hour 0) and run on the plate reader, remove 3 200 uL aliquots from 2-L flask

Hour 8 (4:15)- remove 25 mL from 2-L flask and add salicylic acid, remove 3 200 uL aliquots from Hour 0 and run on the plate reader, remove 3 200 uL aliquots from 2-L flask

Hour 9 (5:15)- remove 3 200 uL aliquots from 2 125-mL flasks (salicylic acid added at Hour 0 and Hour 6) and run on plate reader, remove 3 200 uL aliquots from 2-L flask

Hour 10 (6:15)- remove 25 mL from 2-L flask and add salicylic acid, remove 3 200 uL aliuqots from Hour 0 and run plate reader, remove 3 200 uL aliquots from 2-L flask

Hour 11 (7:15)- remove 3 200 uL aliquots from 125-mL flask (salicylic acid added at Hour 8 and 0) and run on plate reader, remove 3 200 uL aliquots from 2-L flask

Hour 11:15 (7:30)- remove 25 mL from 2-L flask and add salicylic acid, remove 3 200 uL aliquots from 2-L flask

Hour 12 (8:15)- remove 3 200 uL aliquots from 125-mL flask (salicylic acid added at Hour 0) and run on plate reader, remove 3 200 uL aliquots from 2-L flask

Hour 13 (9:15)- remove 3 200 uL aliquots from 125-mL flask (salicylic acid added at Hours 0 and 10) and run on plate reader, remove 3 200 uL alqiuots from 2-L flask

Hour 14:15 (10:30)- remove 3 200 uL aliquots from Hours 0 and 11:15 and run on plate reader, remove 3 200 uL aliquots from 2-L flask

Results

They can be seen in the excel file sent to the grad students. Basically, when salicylic acid is added at the beginning, weird results are found. There is little contrast between J45996 and I7100, and for some odd reason, J45996 produces more GFP in the presence of salicylic acid than in its absence. However, the results from a three-hour pulse with salicylic acid looked very good with GFP continuously going up in I7100 and going down in J45996.

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