IGEM:MIT/2005/Sensor 2: Difference between revisions
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==='''''Done with'''''=== | ==='''''Done with'''''=== | ||
* | *PCR: | ||
#FecA WT | #FecA WT | ||
#FecI | #FecI | ||
#FecR | #FecR | ||
*Biobrick: | |||
#FecA | |||
#FecI | |||
#FecR | |||
*Primers design: | |||
#Adding external linker to scFv | |||
#FecA'-scFv fusion | |||
==='''''Working on'''''=== | ==='''''Working on'''''=== | ||
*PLAN | *PLAN | ||
# | #Checking primers (for: fusion, FecA pro, and adding linker) | ||
# | #Make FecA- -- Work w/ Ray | ||
# | #Make Fur- -- Work w/ Ray | ||
*LAB | *LAB | ||
Mon: digest FecI & FecR miniprep | |||
digest FecA' PCR product | |||
digest | run FecI, FecR, FecA' on gel | ||
gather materials for transformation on Tues. | |||
Tues: transform FecA' | |||
plate FecA' | |||
==Open Issues/Questions== | ==Open Issues/Questions== | ||
*[[../Issues solved/]] | *[[../Issues solved/]] | ||
*[[../Future isses/]] | |||
==Need Help With== | ==Need Help With== | ||
*Need to do: | *Need to do: | ||
# | #Lab: where to get Xgal, IPTG | ||
# | #Check primers --> order through Heather. iGEM account? | ||
*Need opinions/knowledge/expertise: | *Need opinions/knowledge/expertise: | ||
==Experiments== | ==Experiments== | ||
Line 97: | 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> | ||
==='' | ===''Progression''=== | ||
[[../Annie's Notes/]] | |||
'''July Week 3''' | '''July Week 3''' | ||
* | *PCR WT FecA, FecI, and FecR (FecA Promoter failed) | ||
*Biobricked WT FecA | |||
'''July Week 4''' | '''July Week 4''' | ||
* | *Biobricked FecI and FecR | ||
*PCR FecA promoter | *PCR FecA promoter (still failed) --> on hold till new primers come | ||
* | *Started making FecA' and FecR' | ||
'''Aug Week 1''' | '''Aug Week 1''' | ||
'''Aug Week 2''' | '''Aug Week 2''' | ||
==[[../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
Device Parts
- Specified:
- FecA gene BBa_J07017
- FecA' (w/out PstI) BBa_J07018
- FecA promoter BBa_J07019
- FecI gene BBa_J07022
- FecR gene BBa_J07021
- FecR' (w/out PstI) BBa_J07023
- GFP under FecA promoter BBa_J07034
- Test construct for FecA BBa_J07035
- Biobricking: all base components
Current Status
Done with
- PCR:
- FecA WT
- FecI
- FecR
- Biobrick:
- FecA
- FecI
- FecR
- Primers design:
- Adding external linker to scFv
- FecA'-scFv fusion
Working on
- PLAN
- Checking primers (for: fusion, FecA pro, and adding linker)
- Make FecA- -- Work w/ Ray
- 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:
- Lab: where to get Xgal, IPTG
- 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::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.
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