IGEM:Harvard/2007/Quorum Sensing/archives

Week 3

Notes: I have included all four promoters (the R#### parts), but we can just use one or two if we can decide which promoters we don't want. Also, if the FACS appointment is set up Thursday, then that means someone has to grow liquid cultures late in the day on July 4th. The solution is to switch the FACS to Friday and use Thursday to test the transformed cells. Also, the I5311 YFP control looks green under the fluorescent scope in the large room.

• 7/02/07 Monday
  • receive OHHL from Sigma, resuspend, and put in freezer
  • send T9002 and J23039 for sequencing
  • digests:
    • S03623 and S036308 with EcoR1 and Spe1
    • J37034 (we need four samples of this) with Xba1 and Pst1
    • B0015 (we need two samples of this) with EcoR1 and Xba1
    • R0040, R0051, and R0052 with Spe1 and Pst1
  • Set up FACS appointment for Thursday afternoon
• 7/03/07 Tuesday
  • Cip the B0015, R0040, R0051, R0011, R0052 digests
  • Gel extract all the digests that were done on Monday
  • Ligation:
    • S03608+B0015, S03623+B0015, [R0040, R0051, R0011, R0052]+J37034
  • Transform the ligated plasmids into BL21
• 7/04/07 Wednesday
  • 4th of July!
  • grow liquid cultures of T9002, I13263, I13522, I5311 in preparation for the FACS on 7/05/07
  • grow liquid cultures of the transformed ligated parts in preparation for miniprep and sequencing on 7/05/07
• 7/05/07 Thursday
  • FACS with T9002 (tetR promoter, GFP), I13263 (GFP control), I13522 (pL promoter, YFP), I5311 (YFP control)
  • miniprep the transformed ligated liquid culture samples prepared on 7/04/07
  • prepare the miniprepped, transformed, ligated liquid culture samples prepared on 7/04/07 and send for sequencing
• 7/06/07 Friday
  • Find some way to test whether our signal sender constructs actually work. One possibility is to grow up the [promoter]+J37034 and see if there is fluorescence. Are there any ways to detect if our signal senders are creating OHHL?