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

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