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 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

Done with

 * PCR:
 * 1) FecA WT
 * 2) FecI
 * 3) FecR
 * Biobrick:
 * 1) FecA
 * 2) FecI
 * 3) FecR
 * Primers design:
 * 1) Adding external linker to scFv
 * 2) FecA'-scFv fusion

Working on
Mon: digest FecI & FecR miniprep digest FecA' PCR product run FecI, FecR, FecA' on gel gather materials for transformation on Tues. Tues: transform FecA' plate FecA'
 * PLAN
 * 1) Checking primers (for: fusion, FecA pro, and adding linker)
 * 2) Make FecA- -- Work w/ Ray
 * 3) Make Fur- -- Work w/ Ray
 * LAB

Open Issues/Questions

 * ../Issues solved/
 * ../Future isses/

Need Help With

 * Need to do:
 * Lab: where to get Xgal, IPTG
 * 1) Check primers --> order through Heather. iGEM account?
 * Need opinions/knowledge/expertise:

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

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

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.

Progression
../Annie's Notes/ July Week 3 *PCR WT FecA, FecI, and FecR (FecA Promoter failed) *Biobricked WT FecA

July Week 4 *Biobricked FecI and FecR *PCR FecA promoter (still failed) --> on hold till new primers come *Started making FecA' and FecR'

Aug Week 1

Aug Week 2