IGEM:MIT/2005/Receiver 1 experiments: ToxR

Questions

 * 1) Does dimerization initiate a signal?

Experiments

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

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
 * 2) *Test for tags that surround scFv sequence
 * 3) **Flag tag on 3' end of scFv
 * 4) **Use antibodies that recognize those tags; Western, indirect immunofluorescence, permiabilize
 * 5) Location of Fusion Complex: Is it being expressed in correct location (across innermembrane)
 * 6) *Look at cells insitu (microscope), Direct immunofluor.
 * 7) * Fractionate cytoplasmic vs. membrane fractions of cells.
 * 8) *Controls
 * 9) **(-) only ToxR
 * 10) **(-) scFv without tag (we need different primers!)
 * 11) **(+) protein that has flag tag known expressed (need!)
 * 12) Fluorecein Binding to scFv fusion
 * 13) *Fluorescein: When bound to scFv, it does not fluoresce. Cannot test directly. But can lyse cells to get yes/no answer.
 * 14) *Controls
 * 15) **(-) scFv that does not bind fluorecein
 * 16) **(+) scFv that binds fluorecein
 * 17) ***Add fluorecein, Wash cells to remove unbound fluorecein, (+) Should have fluorecein bound to it (-) should not.
 * 18) ***Detect fluorecein (quantitive!)
 * 19) ****(?) Western with antifluorecein rabbit antibody, secondary antibody goat antirabbit (enzyme)
 * 20) ****(?) Pull-down: mix AB with cell solution so that AB attaches to cell, spin down.
 * 21) ****(?) Reverse treatment so that scFv release fluorecein.
 * 22) Does fluorescein dimer initiate dimerization
 * 23) *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)/