IGEM:MIT/2005/Kate's notes from Briefing
Topics that came up...
General Issues to Consider
- What will team define as a successful summer?
- will fusing antibody to protein and testing to see that you got expression be "enough"
- is planning out and presenting a theoretical project "enough"
Antibody signaling group
- Distance between epitopes that would cause transmembrane domains to dimerize?
- Epitopes on the antigen--same or different?
- Can you dimerize the two fusions @ all….are the primary antibodies too big?
- Look @ light receptor from Texas project…Omp system from E. coli?
- Single chain variable regions instead of entire antibody
- Polyclonal antibody instead of monoclonal for the “primary”
- Where to look for sequences of antibodies?
- Antibodies to latex, starch, glycogen, other biological polymers that are unstructured
- Is the metagoal to have a biological sensor?
- Surface expressed/membrane associated antibodies: how do they react to antigen binding? Can we copy that mechanism? Might that allow team to get away from dimerization of the antibody-protein fusions?
- Could we use a yeast pathway that signals in similar ways to a mammalian pathway (like Epo) to get around the issue of moving a pathway from mammalian cells into yeast cells?
- Are their receptor tyrosine kinase pathways in yeast? (Epo signals that way)
- What about bacterial signaling pathways?
- What pathways might be exploited for sensing in yeast?
- mating pathway
- HOG pathway (osmoregulation?)
MAJOR ISSUE: the problem of the yeast cell wall--can't get the antigen or primary antibodies across it!!
Antibody fusion group
- explanation of epitope tags and how they were used in yeast surface display system (Wittrup)
- What is the probability of having an antibody that actually binds to your target of interest?
- Creating and screening a library would take YEARS
- Might you use antibody from the Wittrup lab as a starting point?
- How to fuse the antibody and other protein: how are enzyme linked secondary antibodies made? Are they made as fusion proteins, or by in vitro crosslinking the enzymes and proteins themselves? If fusion protein, then then company should have the sequence encoding the antibodies.
Advisors would like to see by Monday's briefing
- Decision on what system the team will use (yeast, bacteria, etc.) and WHY.
- Decision on what protein will you fuse to for signaling/what signaling pathway will be exploited?