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

	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.