IGEM:MIT/2005/End of Week Meeting: Friday, July 1

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Contents

Notes from End of Week July 1

Division of Labor

  • Lab Plan: Jenny, Jen
  • Antibody: Maxine, Ray, Will
  • Pathway: Jessica, Annie


Action Plan

  • Let's start ordering.
    • Send an e-mail of list of items we need, and then specify of location to obtain them.
  • Design/spec experiments we can do right now.

Antibodies

  • Concern of antirabbit not being expressed - Don't worry about it.
  • Partial cds - did not seq. entirety. (alignment - look for obvious landmarks)
  • Not using antimouse.
  • We need to get the clone, we need the antibody for testing. Call them today!
    • Be careful of evil people.
  • Companies that make scFv's (28 wk w/ antigen, 8 wk w/o), very pricey.
    • Small initial investment for large future gain.
  • Manufacture DNA with fluorecein as part of sequence. Wittrup lab has done this with scFv on yeast.
  • Engr own antigens - add flag to antibody, and antiflag will respond.
  • Fluorecein antibody (rabbit)

Pathways

  • Typical protein assume = 3 nm.
  • ToxR reporter and cI fusion are made. We should order them.


To talk about:

Updates:

  • Applications: we're thinking about it.
  • Antibodies
    • Secondary
      • ScFV antirabbit
not expressed in cell
delete sequence for sensing gold binding
partial cds?
contact author
      • ScFV antimouse-too specific
      • Find backup sequence?
      • Call companies that manufacture ScFVs, contact authors of papers w/scfvs
      • antidigoxin, antidnp; need to check out more. list of commerciall available chem that won't hurt cells?
    • Primary
      • Santra cruz: all bind to specific aa on a human protein: problem?? check w/an advisor
    • Antigens
      • common epitopes: Myc, FLAG (found in sigma aldrich), HA (could not find)
  • Paths we're going down:
    1. ToxR system with small molecules only
    2. ToxR with linker through periplasm to an OMP
      • Build our own linker????
      • Use the MFP (membrane fusion protein) -- use as structural support, etc.
      • Use TonB
    3. Screen FecA linker library
  • Still waiting for word from UCSF, Daniel Kuhne
  • Found Sequences for these coding regions:
    1. ToxR cds
    2. ctx promoter
    3. alkaline phosphotase (used to test for dimerization)
    4. FecA, FecR
    5. TonB
    6. MalE

Questions:

  • Is attempting to use a structural connection between inner and outer membranes proteins insane?
  • Maxine's antibody question
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