IGEM:MIT/2005/Sensor 2

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Revision as of 00:05, 4 August 2005 by Aanniev (Talk)
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Contents

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

Image:FecA.jpg Image: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

  • 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

  • PLAN
  1. Checking primers (for: fusion, FecA pro, and adding linker)
  2. Make FecA- -- Work w/ Ray
  3. Make Fur- -- Work w/ Ray
  • LAB
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'

Open Issues/Questions

Need Help With

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

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::TAGFecA (no scFv)FecA::scFvFecA::TAG/OmpA::TAG
FluorescenceYesNo (-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.

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

Annie's Notes

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