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Project Planning Link

To Do List

Overview

This method for sensing Beta Amyloid relies on an adapted B-Cell Receptor based on the Tango System (LINK). The B-Cell Receptor (Explain How it works here). We will be fusing a TEV protease to Syk and a (TF?????) with a TEV cleavage site to (PROTEIN????). When the Syk is recruited to the receptor the TEV protease will cleave off the (TF????) activating the downstream treatment module.

Experimental Overview

  • Test Antibody Binding

    • Express FP Abeta and lyse cells and wash over beta amyloid?

  • Membrane Localization of all associated Proteins

    • Transfect HEK cells with BCR genes (gantenerumab heavy & light chains, CD79A, CD79B), and Lyn. Tage each component with FP. Check for membrane localization

  • Proper protein Assembly in HEK cells

    • Transfect HEK cells with BCR genes, Lyn, Syk WT & Protein Kinase domain mutation. Culture both Syk types wit hand without AB and ELISA for phosphorylated Syk and/or Lyn ADD FP Fusions

  • Recruitment of Syk to the BCR

    • Transfect HEK cells with BCR genes, Lyn tagged with BFP, Syk tagged with RFP. Culture with and without Beta Amyloid. Check for membrane localization.

  • TEV Protease Cleavage

    • Transfect HEK cells with BCR genes, Lyn, and Syk. Fuse TEV cleavage sites with different fluorescent proteins to the heavy chain, CD79A, and CD79B. Fuse TEV protease to Lyn or Syk. Culture with and without Beta Amyloid and check for membrane delocalization.

  • Full system operation

    • Transfect HEK cells with BCR genes, Lyn, and Syk. Fuse TEV cleavage sites with TF to all/most promising BCR proteins, Fuse TEV protease to Lynor Syk. Transfect TF promoter expressing RFP. Culture with and without Beta Amyloid and check for flourescence. Characterize signal strength based off of flourescence.

Proteins needed

  • CD79A
    • normal
    • fusion: CD79A C terminus + tev cut site + FP
  • CD79B
    • normal
    • fusion: CD79B C terminus + tev cut site + FP
  • Lyn
    • normal
    • fusion: FP tagged (N or C terminus?)
    • point mutation: mutated to always phosphorylate
  • Syk
    • fusion, point mutation: FP attached(N or C terminus?), prevent phosphorylation
    • fusion, point mutatino: tev protease attached (N or C terminus?), point mutated to prevent phosphorylation
  • ig k
    • with proper localization tag
  • igM heavy
    • with proper constant region for membrane localization and proper localization tag

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