IGEM:Harvard/2007/Meetings/Week 7

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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