IGEM:MIT/2005/Receiver 2 experiments: FecA

Make Fusion
1 (a) Fuse FecA with scFv at chosen points and attach chimera to known promoter (makes promoter::FecA::scFv) (b) Make reporter(GFP/lacZ/Tetresistance) with FecA promoter - makes FecApromoter::GFP (c) If needed put FecI and FecR under a known promoter.

Test fecA promoter
2.Transform FecA- strain:
 * FecApromoter::GFP --> Nothing
 * FecApromoter only --> Nothing
 * GFP only --> Nothing
 * Transform wt strain:
 * FecApromoter::GFP --> light up
 * FecApromoter only --> nothing
 * GFP only --> nothing

Test Expression of scFv
3. Transform FecA- strain with 1(a), (b) and (c), or 7 4. Test scFv expression in outer membrane: Detect TAG by Ab*fluorophore --> centrifuge --> FACS

Test overall system
5.Test whether system works: (Negative controls: no GFP under promoter, no scFv, wt FecA strain)

What if it doesn't work?
6. System doesn't work. Troubleshoot. Test binding of antibody with antigen (FRET etc). If ok, then: (a) Either choose a new point to fuse scFv with FecA. Go to 1. (b) Introduce random mutations. Go to 7.

7. Miniprep plasmid DNA and insert random mutations (by either transposons/Drew's paper/circularization). Go to 3.