BCR 2.0

 

Parts Library

Parts Tree

Cloning Progress

Experiments

Timeline

Protocols

 

 

Cloning Strategy
CD79A Linker Optimization
Antibody Choices 

 

Wednesday's To Do's
  • Flow cytometry with Brian (big grin)
  • Analyze flow cytometry data
  • Miniprep and verify, then sequence
    • CD79A-TCS-G4 SDM
    • CD79B-TCS-G4 SDM
  • Midiprep (and verify?)
    • pEXPR TRE:Syk
    • hEF1a:CymR
    • hEF1a-CuO:mKate
    • CMV5-CuO:mKate
  • LRs from Tuesday (that we probably won't have done because LR clonase shortage...)
  • PCRs of mRFP and mIgM constant region for hEF1a:mRFP-CH (constant heavy chain)
  • Order gBlocks for chicken lysozyme and gp120 (once leader sequence situation is resolved...)
  • Order chicken lysozyme and gp120?
Tuesday's To Do's
  • Non-lab to do's
    • Design (or find?) sequencing primers for mIgM CH (constant region)
    • Plan experiment to test gantenerumab binding to beta-amyloid
    • Leader sequences for ordering gBlocks of variable regions?
    • Review sequencing
      • TRE:Syk-pTet:mRFP-TEV
      • TRE:Syk-pTet:mRFP-tTEV
      • pENTR CD79B-TCS-G4
  • Verify
    • hEF1a:eYFP
    • TRE:eYFP
    • TRE:mKate
  • Miniprep and verify
    • CD79A-linker-G4 colonies (3T3, 3T9, 3T15)
    • CD79A-TCS-G4 SDM colony
    • hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
  • Re-transform SDM reactions to hopefully get more colonies to choose from?
  • Pick colonies
    • pEXPR TRE:Syk
    • CD79A-TCS-G4 SDM
    • CD79B-TCS-G4 SDM
  • LR - probably can't do today because no LR clonase
    • EGSH:mKate
    • hEf1a:VgEcR-2A-RXR
    • hEF1a:mKate
    • hEF1a:Gal4VP16 (remember which pDEST)
    • hEF1a:CD79B-TCS-Gal4VP16 (ONLY after sequencing pENTR CD79B-TCS-Gal4VP16)
Monday's To Do's 
  • make BCR enzyme stock
  • digest, gel:
    • pENTR mKate
    • hEF1a:CD79B-TCS-G4
    • hEF1a:Gal4VP16
  • sequence:
  • transform:
    • TRE:syk because we don't have a midi
    • Re-transform CD79A-TCS-G4 and CD79B-TCS-G4 SDM Golden Gates 
  • pick more CD79A linker colonies (all old DNA is not good)
  • pick CD79A SDM colony 
  • pick hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate

 

CMV5-CuO 715:

hEF1a-CuO 715:09

CymR-CuO 710:09


Geneious To Do List:
  • Antibody cloning for chicken lysozyme - Kathryn 
  • hef1a sequencing primers

 

BCR 2.0 To Do

^^^Email: bcrpeeps@mit.edu

^^^Pass: grapefruit

 

 

Higher Level To Do's:
  • Decide on other antigen-variable regions we want to test  
  • Other experimental planning (on experimental page)
  • Parts list (excel sheet detailing specs/locations of parts)
  • How do we do site directed mutagenesis?
  • Literature research: 
    • kozak sequences for heavy and light chains and CD79s
    • truncated TEVp
    • other antigens to detect using BCR (we were thinking of small molecule detection)
    • inducible promoters (we were considering LacI - an inducible repressor - but we want to look into other options first) 
  • Cloning planning 
    • hEF1a:Syk-pTet:mRFP-TEVp
    • variable region 
  • Is gantenerumab proprietary and can we actually publish with it?