• Created by Unknown User (krbrink@mit.edu), last modified by Unknown User (eersland@mit.edu) on Jun 11, 2014 17:21

You are viewing an old version of this page. View the current version.

Compare with Current View Page History

« Previous Version 37 Next »

This is a list of experiments that we would like to do:

 

WhatWhyPartsBasic procedureInput (independent variable being tested)Expected Output (dependent variable being measured, preferably with meaning and failure modes)
Point mutate Syk to keep it from being phosphorylatedNo phosphorylation = no downstream effectsSyk plasmid   
Point mutate Lyn to make it autophosphorylateControl: Even if the BCR complex does not work (eg doesn't detect amyloid), we should still see Syk recruitmentLyn 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)

test 1: CD79A/B and one test for each leader sequence used, stained igM targeting antibody

control: HEK cells, no BCR circuit

test1: outside of cell glows = correct localization

cell doesn't glow = bad localization, failure to express, or antibodies secreted.

control: no glow anywhere

Check IgM binds to beta amyloid ?, antibodydo some kind of a wash step to see if it sticks?  Express antibody, lyse cells and wash over beta amyloid, wash with a dyed IgM targeting antibody.one test for each antibody with dyed antibody, and

control should not glow

Glow should mean that dyed antibody didn't wash off because it stuck to IgM and IgM didn't wash off because it stuck to beta amyloid

lack of glow in test means either the antibody doesn't bind to amyloid or that our test is borked (we could try to control for a bad test by using an antibody that we know works that doesn't bother with the membrane).

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/badd 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-GFPAdd all BCR not FP tagged, add FP tagged Lyn, see what glows

test 1: BCR, lyn

control: no BCR

cell membrane glows: correct localization

Cytosol glows: not binding to CD79

no glow: not being expressed

control: cytosol should glow?

Check Syk localization Syk-GFP, BCR, beta amyloidadd all BCR and get Lyn phosphorylated, and FP tagged Syk

test 1(control): normal Lyn

test 2: autophsosphorlated Lyn

test 1: Syk should float in cytosol

test 2: Syk should localize to membrane

(if test 2 fails, it my be because the localization is too weak to test, so we should use non mutant syk + tev and assay for phosphorylation)

 

Check TEV cleavage on Syk

(and heavy chain)

 

Syk+TEV (variable)

Heavy chain

Light chain

beta-amyloid

Lyn-autophosphorylation and amyloid and anti IgM

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

control: no FP

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 that beta amyloid causes syk recruitment (may be just to debug if the whole system check fails, if whole system check passes, we don't need this) 

Syk-FP

CD79 not tagged

IgM

Lyn, normal

assemble cells, measure Syk localization as a function of beta amloidVary beta amyloid (either continuum or none and a lot or none and a few levels).  Do one test for each leader sequence.Should get a curve of how much syk localizes to the membrane as a function of beta amyloid concentration.
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)

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 (FP tagged SYK shouldn't be in same cell as FP anything else, ought be no cross talk)
    • 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
    we have eBFP(2?), eYFP, mKATE

Supplies needed

  • Antibody that targets membrane bound igM that is dyed so we can see it (we can buy this, probably target heavy chain, may also want light chain as well to make sure both localize)
  • Beta amyloid (? some kind that binds to cells and some kind we can wash antibodies over?)

 

Things to test:

  • does antibody stick to beta amyloid?
  • Membrane localization
    • does antibody get to membrane?
    • does cd79A/B get to membrane?
    • does lyn attach?
    • does syk get recruited by lyn?
    • ? test CD79x dimerization with gel
    • ?test CD79x-IgM attachement with gel
  • does TEV cut anything?
  • does TEV affect Syk localization(assay for phosphorylation of syk)
  • does TEV linked to syk cleave cut correct site?
  • -how much does it cut as a function of amount of Syk?
  • -where should we put molecule to be cleaved?
  • -Does using a transcription factor work (drive an FP), do after finding where to attach
  • does beta amyloid trigger recruitment of syk
  • does beta amyloid trigger release of signal
  • -get an input-output curve for beta amyloid
  • No labels