IGEM:MIT/2005/Sensor 2: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
>Aanniev |
>Aanniev |
||
Line 52: | Line 52: | ||
==Open Issues/Questions== | ==Open Issues/Questions== | ||
#FecA, FecI, and FecR same promoter have issues? | #FecA, FecI, and FecR same promoter have issues? | ||
#Delete loops that's not necessary to make the FecA-scFv work | #Delete loops that's not necessary to make the FecA-scFv work |
Revision as of 23:41, 28 July 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
- Homologous sequence to knock out FecA -- Work w/ Ray
- Obtain Fur- from Boston Medical Center -- POC Ray
- LAB
Fri: digest FecI-BB and FecR-BB to check insertion make freezer stock of FecI-BB and FecR-BB QuikChange on FecA and FecR Next week:
Open Issues/Questions
- FecA, FecI, and FecR same promoter have issues?
- Delete loops that's not necessary to make the FecA-scFv work
- [[../Issues solved/]]
Need Help With
- Need to do:
- [[../Linker for scFv/]]
- Get Fur- -- Work w/ Ray
- 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
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, FecI, and FecR Make FecA', FecR'
July Week 4 *Finish making FecA' and FecR' *PCR FecA promoter *Assemble FecA promoter with GFP
Aug Week 1 *Make FecA'-scFv fusion
Aug Week 2 *Assembling parts