IGEM:MIT/2005/Experiments

[[../Experimental Flowchart/]]

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Experiment Status

Jenny

scFvs: 4M5.3, S101A, 4420
    biobrick ends
    digest scFv + pSB1AK3-1 plasmid w/ E/S 
    gel purify each
    ligate together
    transform
    overnights for miniprep
    identify correct clones (digest)

FecA (full length)
    PCR bb ends
    digest FecA + pSB1AK3-1 plasmid w/ E/S
    gel purify each
    ligate together
    transform
    overnights for miniprep
    identify correct clones

Jessica

MalE
    PCR
    digest MalE + pSB1AK3-1 plasmid w/ E/S 
    gel purify each
    ligate together
    transform
    identify correct clones

PhoA
    PCR with higher annealing temperature
    digest PhoA+ pSB1AK3-1 plasmid w/ E/S 
    gel purify each
    ligate together
    transform
    identify correct clones

Annie

FecA promoter
    PCR with higher annealing temperature
    digest FecApro + pSB1AK3-1 plasmid w/ E/S 
    gel purify each
    ligate together
    transform
    identify correct clones

FecI
    PCR bb end
    digest FecI + pSB1AK3-1 plasmid w/ E/S
    gel purify each
    ligate together
    transform
    identify correct clones

FecR
    PCR bb end
    digest FecR + pSB1AK3-1 plasmid w/ E/S
    gel purify each
    ligate together
    transform
    identify correct clones

All

All
    overnights of bbs, miniprep plasmids
    Grow up overnights (W) and make glycerol freezer stocks (R) -- Jen -- see inventory page for updates

Experimental Timeline

• [[../July wk 3:/]]

1. Receive primers [math]\displaystyle{ \rightarrow }[/math] Make biobricks, with these [[../Techniques/]] 
   J07005 (phoA), J07006 (malE), J07012-14 (scFv 1-3), J07018 (FecA), J07019 (FecA promoter), FecR, FecI.
1.5  Make competent MC4100 cells, pour LB Kan plates, make freezer stocks of strains acquired from Registry
2. ToxR: 
   Can begin assembly of R0040::B0030, for use in parts J07024-28 (see August wk. 1)
3. Input Experiments : Verify that flourescin oligo's enter cells. 
4. Assemble fecA promoter with GFP --> test (after J07019 complete)

• July wk 4:

FecA: Fuse scFv with FecA?
ToxR:

• Aug wk 1:

ToxR:
 -Receive synthesized J07007 (ctx promoter), J07009 (ToxR')
 -Begin Assembly of: 
   -J07011 (ctx promoter :: gfp reporter) 
   -J07024-26 (promoter::RBS::ToxR'::scFv 1-3)
   -J07027-28 (Positive and Negative controls, promoter::RBS::ToxR'::PhoA/MalE)

• Aug wk 2:

• Aug wk 3:

• Aug wk 4:

• Sept wk 1