IGEM:MIT/2005/Experiments

[[../Experimental Flowchart/]]

 indicates done!
 indicates pending...

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)
    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
    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

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

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

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