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Results from Sequencing

Both J45120 and J45181 looked good, although J45181 had a three base pair addition late in the protein's coding region. However, that is certainly unrelated to the puzzling results collected from the time course. The osmY promoter (and inverter) structures for J45995 and J45996 (fluorescent phase-sensitive devices) are identical to J45181's structures. After talking with Barry, it seems like a good next step to run protein gels at various time points for J45120 and J45181 to confirm the GC/smell results.

Results from Plate Reader Experiment

As with other runs, J45995, J45996, B0015, and the plain media wells behaved as predicted in a consistent manner with other results. However, R0040.E0840 gave fluorescent levels five to ten times greater than the other devices. At this point, it seems that we really need to narrow down why R0040.E0840 is giving varying results. After consulting with Barry, we have decided to:

  • Make fresh plates for each part before the plate reader experiment and grow them each up for the same durations
  • Make fresh LCs from single colonies from the fresh streaks and only grow them up for 10 hrs (since we suspect that varying buildup of GFP in R0040.E0840 in stationary phase could be the culprit for variation in the data)
  • Measure OD600s for those overnight LCs
  • Compensate for the OD600s when diluting into media and growing for 50 mins

Other Stuff to Do

1. Talk with Dr. Knight about media (1:30ish to 2:00ish)- DONE (will try M9 Minimal Media + M9 Minimal Media with proline, aspartic acid, alanine, and serine)

2. Talk with Drew about abstract/general project

3. Restreak J45995, J45996, R0040.E0840, and B0015 (just before leaving)

4. Contact Alex Bradley about GC/MS run- DONE

5. Ask Meagan about IK J45120 and J45200- DONE (pick up plates ~5ish)- DONE (growing up a plate for overnight)

6. Make M9 Minimal Media

  • 500 mL 2X M9 Salts
  • 429.8 mL H20 (anticipate 20 mL 20% carbon source)
  • 20 mL 0.1 M MgSO4 (2 mM MgSO4)
  • 200 uL 0.5 M CaCl2 (.1 mM CaCl2)
  • 30 mL 10 mg/mL thiamine hydrochloride

Split in half to 490 mL.

To one of the 490 mL stock added:

  • 0.71 g proline
  • 0.57 g aspartic acid
  • 0.196 g alanine
  • 0.3275 g serine
  • ALL are half what was called for in the liter stock for defined medium
  • Plus autoclave for 20 mins on fluid cycle
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