IGEM:MIT/2006/Notebook/2007-8-14

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Smell Course Set Up

Protocol for 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

Today's experiment

Time Zero= 10:53 (time at which master flask starts shaking at 110 RPM, wanted to do it earlier but the flask broke on the shaker and spent a good hour or so fixing it and then needed to redo cultures)

Hour 5= 3:53 (add salicylic acid to a culture)

Hour 6= 4:53 (add salicylic acid to a culture)

Hour 8= 6:53 (smell Hour 5 culture)

  • J45120- 1.44, J45181- 1.52
  • Arun- J45120 smells more
  • Matt- J45120 stinks worse
  • Francois- J45181 smells barely more
  • Tony- J45181 smells more
  • Stephen- J45181 smells barely more

Hour 9= 7:53 (smell Hour 6 culture)

  • J45120- 1.86, J45181- 1.90
  • Stephen- J45181 smells more
  • Barry- pretty similar, maybe J45181 smells slightly more

Hour 10.5= 9:23 (add salicylic acid to a culture)

Hour 11.5= 10:23 (add salicylic acid to a culture)

Hour 13.5= 12:23 (smell Hour 10.5 culture)

  • J45120- 2.08, J45181- 2.18
  • Stephen- J45181 smells slightly more (both really strong though)
  • Jason- they smell the same

Hour 14.5= 1:23 (smell Hour 11 culture)

  • J4120- 2.26, J45181- 2.28
  • Stephen- J45120 smells slightly more (both really strong though)
  • Jason- J45120 smells more

Conclusion- will go ahead and try the experiment

Other Things to Do Today

1. Meeting with Alex at 1 PM to obtain column and protocol- DONE (OBTAINED)

2. Add PCNB to GC samples from yesterday- DONE

3. Run the GC samples from yesterday to estimate GC extraction efficiency/MS decay rate- DONE

  • Unfortunately, as suspected the small amount of methyl salicylate added to the water must have not diffused about but rather stayed as a droplet. Thus, the efficiency samples came out extremely low
  • However, the sample which I added straight methyl salicylate to and grew up the culture for two additional hours did come out okay. I added 16 ppm, or .429 uL of the pure stuff to a 25-mL culture. I got a yield of 113.8 ppm. Thus, my efficiency was 113.8 ppm/10/16 ppm = 71.2%. Preliminary extraction data from before was ~70-80%. Thus, methyl salicylate does not degrade significantly after 2 hours; thus, my time course from last week was not the ideal protocol. Hopefully, the 3-hour pulse will work.

4. At 6:45, put in LCs of J45995, J45996, B0015, and R0040.E0840

  • Plan- Grow up LCs for 17 hours, Dilute 1:250 and grow up 25-mL cultures in 250-mL flasks at 220 RPM at 37C, Dilute 1:25 and grow up for 1:15

5. Check sequencing of J45250 1AT3- DONE (looked good, had the same point mutation as J45250 3K3 in coding region of ATF1, which means that it is extremely likely that the original J45250 1AC3 had the same point mutation as well)

6. Unpack new load of heptane- DONE

7. Made LC of J45250 transformant after realizing that I stupidly threw the LC correctly sequenced away during cleaning- DONE

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