IGEM:MIT/2005/Experiments

[[../Experimental Flowchart/]]

 indicates done!
 indicates pending...

Experiment Status

Jenny

scFv.TT (J07012-14.B0015)
    digest B0015 E/X, recipient
    obtain digested scFvs E/S, prefix donor
    gel purify B0015
    ligate J07012-14.B0015
    transform
    overnights for miniprep
    identify correct clones (digest), streak plates
    overnights for freezer stock
    freezer stocks scFv.B0015
   

Promoter.RBS.scFv
    digest Promoter.RBS BB (recipient)
    obtain digested scFvs (suffix donor)
    gel purify promoter.RBS
    ligate promoter.RBS.svFv
    transform
    overnights for miniprep
    identify correct clones (digest), streak plates
    overnights for freezer stock
    freezer stocks promoter.RBS.scFv
   

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)
    overnights for freezer stock
    freezer stocks

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
    overnights for freezer stock
    freezer stocks

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

PhoA'
    QuikChange

Annie

FecA promoter
    PCR with current primers with higher annealing temperature
    PCR with newly designed primers
    digest FecApro + pSB1AK3-1 plasmid w/ E/S 
    gel purify each
    ligate together
    transform
    identify correct clones (digest)
    freezer stock

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

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

FecA'
    QuikChange

FecR'
    QuikChange

Project Experiments

  Input Device: [[../Input: Ligand#Experiments/|fluorescein]]
  Head Receiver Unit: [[../Receiver Head Unit: scFv#Experiments/|anti-fluorescein scFv]]
  Receiver 1: [[../Receiver 1: ToxR#Experiments/|ToxR]]
  Receiver 2: [[../Sensor 2#Experiments/|FecA]] 
  Signal Processor: [[../Signal processing#Experiments/|Exp. Here]]
  Actuator: [[../Actuator#Experiments/|Exp. Here]]

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