IGEM:MIT/2005/Receiver 1 experiments: ToxR

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Receiver 1 w/out Head Unit

Questions

  1. Does dimerization initiate a signal?

Experiments

  1. Does dimerization initiate a signal
    • Requires testing of ToxR::PhoA and ToxR::MalE, with ctx promoter::actuator.

Receiver 1 w/ Receiver Head Unit

Questions

  1. Is the fusion complex being expressed?
  2. Is the protein where we want it to be?
  3. If we make fusion to ToxR, can scFV bind to fluorecein?
  4. Will the binding of a fluorescein dimer initiate dimerization?

Experiments

  1. Expression of Fusion Complex
    • Test for tags that surround scFv sequence
      • Flag tag on 3' end of scFv
      • Use antibodies that recognize those tags; Western, indirect immunofluorescence, permiabilize
  2. Location of Fusion Complex: Is it being expressed in correct location (across innermembrane)
    • Look at cells insitu (microscope), Direct immunofluor.
    • Fractionate cytoplasmic vs. membrane fractions of cells.
    • Controls
      • (-) only ToxR
      • (-) scFv without tag (we need different primers!)
      • (+) protein that has flag tag known expressed (need!)
  3. Fluorecein Binding to scFv fusion
    • Fluorescein: When bound to scFv, it does not fluoresce. Cannot test directly. But can lyse cells to get yes/no answer.
    • Controls
      • (-) scFv that does not bind fluorecein
      • (+) scFv that binds fluorecein
        • Add fluorecein, Wash cells to remove unbound fluorecein, (+) Should have fluorecein bound to it (-) should not.
        • Detect fluorecein (quantitive!)
          • (?) Western with antifluorecein rabbit antibody, secondary antibody goat antirabbit (enzyme)
          • (?) Pull-down: mix AB with cell solution so that AB attaches to cell, spin down.
          • (?) Reverse treatment so that scFv release fluorecein.
  4. Does fluorescein dimer initiate dimerization
    • Can we separate out dimerization from signal generation?


[[../Tabled because we do not have ToxR-cI fusion: Signal Test (see: John Mekalanos, Harvard, ToxR-cI Fusion)/]]