IGEM:Harvard/2007/Meetings/Week 7

Perry's presentation, 7-30-07 (Powerpoint 2007)

Perry's presentation 7-30-07, compatible with older versions of Powerpoint

Perry's drop experiment animation (Powerpoint 2007)

Perry's drop experiment animation, compatible with older versions of Powerpoint

Stephanie's slide

Sambu's ppt (Powerpoint 2007)

George's presentation, 7-30-07

S.Lo's Notes

• Perry
  • GFP-mek/PDZ cell binding assay: No significant fluorescence
  • In vitro assay
    • Glutathione bead used
    • Scale of original invitro assay not great (485/538, quorum-sensing protocol)
    • Results not clear when using different pair of wavelengths (for EGFP)
  • Quorum
    • Drop Experiment
      • Plate Receiver, lawns
      • Add Red OHHL sender into middle of agar plate
      • Green halo seen around center
    • Rebuilding Voigt construct
      • lux R and lux I controlled by lux pR promoters, GFP also controlled by pR promoter
      • Perry building through PCR/digest assembly
        • Worked until attempt to put together luxR-luxpR promoter and B0034-luxI - 2kb fragment disappeared - perhaps forward primer just binds to luxI and amplifies just luxI end part
      • Question: why need to rebuild this construct? Answer: more "offness" of construct
      • Alain's comment: Voigt paper has luxI and luxR in opposite directions; play with Biobrick sites and make it backwards
      • Harris - ask Voigt for construct? (George will email)
• George
  • Switched fluorophores
    • GFP/RFP folding/maturation assay?
    • Growth, not induction, of T02 (small range of GFP, comparable to tetR GFP sender)
  • FACS
    • Overnight cultures, serially diluted (1:10, 1:100, 1:1000, 1:10000)
    • Measured OD before appt (only grew up for a few hrs)
    • Wanted to see lower concentrations (below quorum) w/ mean GFP per OD lower than quorum-reaching dilutions
    • Redo with JT diluted multiple times, watch percent of total cells fluorescent
    • Go to OD that's higher (compare/contrast quorum-reaching)
• Shaunak
  • Refer to ppt
• Sammy
  • Refer to ppt
  • Preliminary results look good, ie results from two-step PCR and gel are as expected
• Kevin
  • Refer to "Plates" subpage of Wiki; colony counts of MACS
  • Allow selection of white colonies, only a few red
  • Flow cytometry: hard to find difference between negative and positive
    • Second time - red, no white, colonies
    • Fluorescent tags not compatible with wavelength?
  • Outlook: Run another MACS this week, try to get selection
    • Use a background that is not fluorescent (lacZ?)
    • Try MACS, purifying, then going to FACS
    • Focus on inserting library
• Alex
  • Miniprep did not work