IGEM:MIT/2005/Receiver 1 experiments: ToxR
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Receiver 1 w/out Head Unit
Questions
- Does dimerization initiate a signal?
Experiments
- Does dimerization initiate a signal
- Requires testing of ToxR::PhoA and ToxR::MalE, with ctx promoter::actuator.
Receiver 1 w/ Receiver Head Unit
Questions
- Is the fusion complex being expressed?
- Is the protein where we want it to be?
- If we make fusion to ToxR, can scFV bind to fluorecein?
- Will the binding of a fluorescein dimer initiate dimerization?
Experiments
- 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
- Test for tags that surround scFv sequence
- 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!)
- 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.
- 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)/]]