IGEM:MIT/2005/Sensor 2

POC

FecAnnie

Office: BUG Lounge

Email: aanniev@mit.edu

Phone: 617-252-1680

X5-6256 (dorm)

Function

• Main component: FecA protein
• Purpose: fusion with scFv
• Mechanism: ligand binds to scFv induces conformational changes in FecA protein --> PoPS

Device Depiction

File:Receiver.2.FecA.ppt

Device Parts

• Specified:

1. FecA gene BBa_J07017
2. FecA' (w/out PstI) BBa_J07018
3. FecA promoter BBa_J07019
4. FecI gene BBa_J07022
5. FecR gene BBa_J07021
6. FecR' (w/out PstI) BBa_J07023
7. GFP under FecA promoter BBa_J07034
8. Test construct for FecA BBa_J07035

• Biobricking: all base components

Current Status

Done with

• Primers:

1. native FecA
2. to point-mutate FecA
3. FecA promoter
4. FecI
5. FecR
6. to point-mutate FecR

• Experiment read out
• Cells:

1. FecA+ (MC4100)

Working on

• Redesign FecA promoter primers
• Work out gene sequences and other materials for internal fusion
• Spec out FecA-scFv
• Make our own FecA-
• Obtain Fur- from Boston Medical Center
• Specifying composite parts

Open Issues/Questions

1. There are two ways to make FecA-: recombineering and gene replacement using pKO vector.
2. FecA, FecI, and FecR same promoter have issues?
3. Delete loops that's not necessary to make the FecA-scFv work

• [[../Issues solved/]]

Need Help With

• Need to do:

1. Internal fusion
2. Get Fur- -- Work w/ Ray
3. Make FecA-

• Need opinions/knowledge/expertise:

1. FecA, FecI, and FecR relationship w/ respect to control of one promoter
2. Alternative methods for gene fusion: random mutation by circular permutation and XL1-Red

Experiments

META LEVEL

1.Build

(a) [[../Fuse FecA with scFv/]] at chosen points and attach chimera to known promoter (makes promoter::FecA::scFv)

(b) Attach FecA promoter with reporter(GFP) - makes FecApromoter::GFP

(c) If needed put FecI and FecR under a known promoter.

2.Tests Test expression of (b):

• 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

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)

5.Test overall system: (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.

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.

TIME SCHEDULE

July Week 3
   *Make stock of WT FecA, FecA promoter, FecI, and FecR
    Make FecA' 
   *Assemble FecA promoter with GFP
   *Order: primers for gene sequences for fusion 
   *Obtain: Fur-

July Week 4 
   *Make FecR' 
    Make FecA-
   *Make FecA-scFv fusions

Aug Week 1
   *Finish making FecA-scFv fusions
   *Assemble parts

Aug Week 2
   *Finish assembling parts

Aug Week 3

Aug Week 4