IGEM:MIT/2005/Sensor 2: Difference between revisions

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>Aanniev
 
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==='''''Done with'''''===
==='''''Done with'''''===
*Primers:  
*PCR:
#native FecA
#FecA WT
#to point-mutate FecA
#FecI
#FecA promoter
#FecR
*Biobrick:
#FecA
#FecI
#FecI
#FecR
#FecR
#to point-mutate FecR
*Primers design:
*Experiment read out
#Adding external linker to scFv
*Cells:
#FecA'-scFv fusion
#FecA+  (MC4100)
 
==='''''Working on'''''===
==='''''Working on'''''===
*Redesign FecA promoter primers
*PLAN
*Work out gene sequences and other materials for internal fusion
#Checking primers (for: fusion, FecA pro, and adding linker)
*Spec out FecA-scFv
#Make FecA- -- Work w/ Ray
*Make our own FecA-
#Make Fur- -- Work w/ Ray
*Obtain Fur- from Boston Medical Center
*LAB
*Specifying composite parts
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==
==Open Issues/Questions==
#There are two ways to make FecA-: recombineering and gene replacement using pKO vector.
#FecA, FecI, and FecR same promoter have issues?
#Delete loops that's not necessary to make the FecA-scFv work
*[[../Issues solved/]]
*[[../Issues solved/]]
*[[../Future isses/]]


==Need Help With==
==Need Help With==
*Need to do:
*Need to do:
#Internal fusion
#Lab: where to get Xgal, IPTG
#Get Fur- -- Work w/ Ray
#Check primers --> order through Heather. iGEM account?
#Make FecA-
*Need opinions/knowledge/expertise:
*Need opinions/knowledge/expertise:
#FecA, FecI, and FecR relationship w/ respect to control of one promoter
#Alternative methods for gene fusion: random mutation by circular permutation and XL1-Red


==Experiments==
==Experiments==
Line 99: Line 99:


<b>7.</b> Miniprep plasmid DNA and insert random mutations (by either transposons/Drew's paper/circularization). Go to <b>3.</b>
<b>7.</b> Miniprep plasmid DNA and insert random mutations (by either transposons/Drew's paper/circularization). Go to <b>3.</b>
===''TIME SCHEDULE''===
===''Progression''===
[[../Annie's Notes/]]
  '''July Week 3'''
  '''July Week 3'''
     *Make stock of WT FecA, FecA promoter, FecI, and FecR
     *PCR WT FecA, FecI, and FecR (FecA Promoter failed)
    Make FecA'
     *Biobricked WT FecA
     *Assemble FecA promoter with GFP
    *Order: primers for gene sequences for fusion
    *Obtain: Fur-


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


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


  '''Aug Week 2'''
  '''Aug Week 2'''
    *Finish assembling parts
'''Aug Week 3'''
'''Aug Week 4'''


==[[../Annie's Notes/]]==
==[[../Annie's Notes/]]==

Latest revision as of 21:05, 3 August 2005

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

  • 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

  • [[../Issues solved/]]
  • [[../Future isses/]]

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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