This is a list of experiments that we would like to do:
| What | Why | Parts | Basic procedure | Input | Output |
|---|---|---|---|---|---|
| Point mutate Syk to keep it from being phosphorylated | No phosphorylation = no downstream effects | Syk plasmid | |||
| Point mutate Lyn to make it autophosphorylate | Control: Even if the BCR complex does not work (eg doesn't detect amyloid), we should still see Syk recruitment | Lyn plasmid | |||
Check IgM localization using cytometry (see Hombach paper) (is this needed, if CD79 localizes, we are pretty sure IgM had to localize correctly) | IgMkappa IgM heavy CD79a CD79b | add all CD79A, CD79B, heavy chain, light chain all untagged. Add stained antibody that targets igM (heavy and/or light?) and see if it sticks to the cell membrane. (add and wash) | CD79A/B and one test for each leader sequence used, stained igM targeting antibody | outside of cell glows = correct localization cell doesn't glow = bad localization, failure to express, or antibodies secreted. | |
| Check CD79a/b localization (we have to have a CD79 fused to an FP for the final product, so might as well test localization now) | fluorescently tagged CD79a/b | add all BCR components, with one CD79 at a time in its FP tagged version to check correct localization | test 1: CD79A-tev-FP, CD79B, igM test 2: CD79B-tev-FP, CD79A, igM | test 1: cell membrane glows: correct localization. Cytosol glows = not membrane localized. No glow = not produced test 2: same, but different tested protein | |
| Check Lyn localization | Lyn-GFP | Add all BCR not FP tagged, add FP tagged Lyn, see what glows | test 1: BCR, lyn | cell membrane glows: correct localization Cytosol glows: not binding to CD79 no glow: not being expressed | |
| Check Syk localization | Syk-GFP | add all BCR and Lyn that is point mutated to always be phosphorylated, and FP tagged Syk | test 1: no Lyn test 2: autophsosphorlated Lyn | test 1: Syk should float in cytosol test 2: Syk should localize to membrane | |
| Check TEV cleavage | Syk+TEV (variable) Heavy chain Light chain
Lyn-autophosphorylated CD79-TEVsite-GFP (see if CD79A, CD79B, or combination is better) | add all components and a varying amount of syk+tev on an inducible promoter to amount of cleavage. Should have an FP tied to syk+tev to measure amount of syk_tev floating around | test 1:CD79a-TEV-FP test 2:CD79b-TEV-FP test 3:CD79a-TEV-FP and CD79b-TEV-FP | test 1: should make a curve of how much FP is cleaved as a function of syk+tev concentration test 2: same test 3: Output will be FP from CD79A and CD79b combined as a function of syk+tev | |
| Check whole system | CD79-TEV-FP igM heavy and light Lyn Syk-TEV beta amyloid (variable) | add CD79A/B (with FP tag from tev cleavage test), Lyn, syk+TEV, and one test for every leader sequence that works. Add some amount of beta amyloid and get a curve of FP cleavage to beta amyloid (or, if knowing beta amyloid concentration is hard, we should still characterize something so that future researchers can tune the amount of output). | for each antibody leader: add components, add beta amyloid (there should be a control with 0 beta amyloid, I'd like to have varying amounts of beta amyloid) | Some amount of FP should move from membrane to cytosol (either compare to control of no amyloid or compare intensity of membrane to intensity of cytosol) |
does antibody get to membrane?
does cd79A/B get to membrane?
does lyn attach?
does syk get recruited by lyn?
does TEV cleave cut site?
does TEV linked to syk cleave cut site?
-how much does it cut as a function of amount of Syk?
-where should we put molecule to be cleaved?
does beta amyloid trigger release of signal
-get an input-output curve for beta amyloid