IGEM:MIT/2005/Receiver 2 experiments: FecA
Experiments
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
| FecA::scFv::TAG | FecA (no scFv) | FecA::scFv | FecA::TAG/OmpA::TAG | |
|---|---|---|---|---|
| Fluorescence | Yes | No (-ve control) | No (-ve control) | No (+ve control) |
Test overall system
5.Test whether system works: (Negative controls: no GFP under promoter, no scFv, wt FecA strain)
| + Fluorescein | - Fluorescein | To Do Next |
|---|---|---|
| GFP produced | No GFP produced | System works!! --> Characterize system |
| Anything else | Anything else | Go To 6. |
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.
