Project Planning

To Do

• Further BCR Research
  •  How is Lyn Phosphorylated?
  • Is Syk Recruited Before Abeta binding
  • Which Parts of the BCR proteins are extracellular/Intracellular
    cd79's: N-signal peptide-extracellular domain-helical transmembrane-cytoplasmic-C
  • Switch TEV cleavage site and TEV Protease
    doesn't work because of nuclear localization
  • Why don't antibodies start with a Met (use self cleaving protein?)
    • Turns out that the first few amino acids tell the cell that this will be put in the membrane and this signal tag is cleaved as part of post translation modification.
    • Find this sequence for heavy and light chain
      • verify function of our sequence
        • find someone who is expressing membrane bound antibodies
        • check immunotherapy, perhaps cancer?
  • Find Antibody Plasmid (multiple)
  • Immunologist interview
  • find all n and C termini on all proteins to know where to attach things
    • syk
      
      • near active site?
    • lyn
      • near active site?
    • heavy
      n is in variable region, c is in membrane
    • light chain
      n is in variable region, c is in constant
    • cd79a
      C terminus inside cell, n terminus outside and contains a membrane localization tag
      C terminus near but separate from immunoreceptor tyrosine-based activation motif(ITAM) by ~30 amino acids
    • cd79b
      same as cd79a
• Lab work
  • Multiply plasmids
    • Syk
    • Lyn
  • Verify? (sequence/gel)
• Experiments
• Planning
  • add BCR steps
• WIki stuff
  
  • Make Research Page
  • use cloaks to make this list collapsible

No point mutating Syk because we’re not going to worry about cross-talk

Talk to Nelson about maximum number plasmids for transfection

Neuronal crosstalk?

TEV cleavage site high or low affinity?

Experiment

-          Western blot HEK293 cells for Syk

• anti-Syk antibody

-          Check BCR membrane localization

• add CD79a/b + mIgM and verify with fluorescent anti-IgM antibodies

-          Check IgM binding

• biotinylated beta-amyloid with fluorescent secondary antibody

-          Check Lyn and Syk membrane localization using fluorescent fusions

• Lyn-YFP
• Syk-mKate

-          Make Syk+TEVp fusions ->   PRIORITIZE

• Fusions
  • N terminus: 0 linking aa
  • N terminus: 15 linking aa
  • C terminus: 0 linking aa
  • C terminus: 15 linking aa
• Inducible expression under TetR

High Priority Fusions

-          Syk-BFP

-          Lyn-mKATE

-          CD79A-TCS-Gal4VP16

-          CD79B-TCS-Gal4VP16

-          Syk-15-TEVp

-          (Syk-0-TEVp)

Use ENLYFQL cleavage site -> best on/off ratio

Add to C terminus of Syk like they did in paper with EGFP

Add to C terminus of Lyn because N terminus in membrane

Pressing tab anywhere in a line moves it right one level, (shift+tab) anywhere in a line moves it left one level.