IGEM:MIT/2005/Experiments: Difference between revisions

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==Experiment Status==
==Experiment Status==


===Jenny===
 
  '''scFvs: 4M5.3, S101A, 4420'''
  '''Stock-up MiniPreps'''
     [[Image:Check.gif]] biobrick ends
     [[Image:Check.gif]] grow up B0015, J13002, E0840
     [[Image:Check.gif]] digest scFv + pSB1AK3-1 plasmid w/ E/S
     [[Image:Check.gif]] overnight B0015, J13002, E0840
     [[Image:Check.gif]] gel purify each
     [[Image:Check.gif]] miniprep B0015, J13002, E0840
     [[Image:Check.gif]] ligate together
     [[Image:Check.gif]] glycerol stock J13002
    [[Image:Check.gif]] transform
 
    [[Image:Pending.gif]] overnights for miniprep
    [[Image:Pending.gif]] identify correct clones (digest)
    [[Image:Pending.gif]] freezer stocks


  '''FecA (full length)'''
  '''FecA (full length)'''
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     [[Image:Check.gif]] ligate together
     [[Image:Check.gif]] ligate together
     [[Image:Check.gif]] transform
     [[Image:Check.gif]] transform
     [[Image:Pending.gif]] overnights for miniprep
     [[Image:Check.gif]] overnights for miniprep
     [[Image:Pending.gif]] identify correct clones
     [[Image:Check.gif]] identify correct clones
     [[Image:Pending.gif]] freezer stocks
     [[Image:Check.gif]] overnights for freezer stock
    [[Image:Check.gif]] freezer stocks


===Jessica===
===Jessica===
  '''MalE'''
  '''MalE'''
     [[Image:Check.gif]] PCR
     [[Image:Check.gif]] PCR
     [[Image:Pending.gif]] digest MalE + pSB1AK3-1 plasmid w/ E/S  
     [[Image:Check.gif]] digest MalE + pSB1AK3-1 plasmid w/ E/S  
     [[Image:Pending.gif]] gel purify each
     [[Image:Check.gif]] gel purify each
     [[Image:Pending.gif]] ligate together
     [[Image:Check.gif]] ligate together
     [[Image:Pending.gif]] transform
     [[Image:Pending.gif]] transform
     [[Image:Pending.gif]] identify correct clones
     [[Image:Pending.gif]] identify correct clones


  '''PhoA'''
  '''PhoA'''
     [[Image:Pending.gif]] PCR with higher annealing temperature
     [[Image:Check.gif]] PCR with higher annealing temperature
     [[Image:Pending.gif]] digest PhoA+ pSB1AK3-1 plasmid w/ E/S  
     [[Image:Check.gif]] digest PhoA+ pSB1AK3-1 plasmid w/ E/S  
     [[Image:Pending.gif]] gel purify each
     [[Image:Check.gif]] gel purify each
     [[Image:Pending.gif]] ligate together
     [[Image:Check.gif]] ligate together
     [[Image:Pending.gif]] transform
     [[Image:Pending.gif]] transform
     [[Image:Pending.gif]] identify correct clones
     [[Image:Pending.gif]] identify correct clones
'''PhoA''''
    [[Image:Pending.gif]] QuikChange


===Annie===
===Annie===
Line 52: Line 53:
     [[Image:Pending.gif]] ligate together
     [[Image:Pending.gif]] ligate together
     [[Image:Pending.gif]] transform
     [[Image:Pending.gif]] transform
     [[Image:Pending.gif]] identify correct clones
     [[Image:Pending.gif]] identify correct clones (digest)
    [[Image:Pending.gif]] freezer stock


  '''FecI'''
  '''FecI'''
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     [[Image:Check.gif]] digest FecI + pSB1AK3-1 plasmid w/ E/S
     [[Image:Check.gif]] digest FecI + pSB1AK3-1 plasmid w/ E/S
     [[Image:Check.gif]] gel purify each
     [[Image:Check.gif]] gel purify each
     [[Image:Pending.gif]] ligate together
     [[Image:Check.gif]] ligate together
     [[Image:Pending.gif]] transform
     [[Image:Check.gif]] transform
     [[Image:Pending.gif]] identify correct clones
     [[Image:Pending.gif]] identify correct clones (digest)
    [[Image:Pending.gif]] freezer stock


  '''FecR'''
  '''FecR'''
Line 66: Line 69:
     [[Image:Check.gif]] digest FecR + pSB1AK3-1 plasmid w/ E/S
     [[Image:Check.gif]] digest FecR + pSB1AK3-1 plasmid w/ E/S
     [[Image:Check.gif]] gel purify each
     [[Image:Check.gif]] gel purify each
     [[Image:Pending.gif]] ligate together
     [[Image:Check.gif]] ligate together
     [[Image:Pending.gif]] transform
     [[Image:Check.gif]] transform
     [[Image:Pending.gif]] identify correct clones
     [[Image:Pending.gif]] identify correct clones (digest)
    [[Image:Pending.gif]] freezer stock
 
'''FecA''''
    [[Image:Pending.gif]] QuikChange
 
'''FecR''''
    [[Image:Pending.gif]] QuikChange


==Project Experiments==
==Project Experiments==
Line 78: Line 88:
   '''Actuator''': [[../Actuator#Experiments/|Exp. Here]]
   '''Actuator''': [[../Actuator#Experiments/|Exp. Here]]


==Experimental Timeline==
[[../Experimental Timeline/]]
*<b>[[../July wk 3:/]]</b>
1. Receive primers <math>\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)
 
*<b>July wk 4:</b>
FecA: Fuse scFv with FecA?
ToxR:
 
*<b>Aug wk 1:</b>
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)
 
*<b>Aug wk 2:</b>
 
*<b>Aug wk 3:</b>
 
*<b>Aug wk 4:</b>
 
*<b>Sept wk 1

Latest revision as of 05:34, 15 August 2005

[[../Experimental Flowchart/]]

 indicates done!
 indicates pending...

Experiment Status

Stock-up MiniPreps
    grow up B0015, J13002, E0840
    overnight B0015, J13002, E0840
    miniprep B0015, J13002, E0840
    glycerol stock J13002



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/]]

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