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Overview B-cell receptors (BCRs) are unique in their ability to recognize a highly specific antigen and produce an intracellular response. Centering around a membrane-bound IgM antibody which performs the actual antigen detection, they also contain a number of helper proteins, in particular the membrane proteins CD79A and CD79B; inside the cell, protein kinases such as Lyn attach to their intracellular domains to receive any signal it transmits. For this project, we plan to engineer a BCR to respond to the beta-amyloid plaques that are the hallmark of Alzheimer's disease. Normally, when an antigen binds to the IgM part of the BCR, CD79A and CD79B become phosphorylated with the help of Lyn, thus recruiting another protein kinase known as Syk. In B cells, this leads to a signaling cascade which induces cell proliferation. In our circuit, we would like the binding of beta-amyloid to transmit a signal in the form of a transcription factor to our diagnosis/treatment module - although this cuts out the signal amplification resulting from the kinase cascade, it leads to a far more controlled and quantifiable cellular response. Our solution involves TEV protease, which cleaves a particular amino acid sequence (TEV cleavage site) that can be used as a linker in fusion proteins. In our system, CD79A and CD79B are linked to transcription factors by TEV cleavage sites and Syk is fused to a TEV protease. When Syk is recruited to the BCR upon beta-amyloid binding, the attached TEV protease will come in close proximity to its cleavage site, where it can cut and release the attached transcription factor into the cell. This transcription factor can then go on to activate the genes involved in our diagnosis/treatment module. Additionally, by a simple substitution our modified BCR could be adapted to detect any antigen (and release any transcription factor), making it a particularly useful synthetic biology tool. |
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Proposed To do list
| To Do |
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| Lab work (Can do)(Waiting on parts)(Dependent on other process) | Experiments | Planning (research?) | To Do Tomorrow |
| |
| - design expression vectors
- make wiki templates
- order antibodies
- make sure we've got any
tags, dyes, etc. - anti-IgM
- anti-Syk
- anti-Lyn
- anti-primary (for blots)
- anti-pSyk (Tyr323)
- update to-do list
- Plan Gibson for BCR Plasmids
| - Sunday-------------------------------------------------------------------------------------------------------------
- Transform LRs
- Pick CD79B-STOP colonies
- Pick Syk-eYFP and Lyn Colonies
- Saturday----------------------------------------------------------------------------------------------------------
- Miniprep Gmab Heavy
- Sequence Gmab Heavy
- Screen CD79B-STOP
- Transform CD79B-STOPs from screen
- Transform stuff from Syk-eYFP and Lyn Screens
- Run LRs
- Design additional alternative cloning primers?????
- Mediprep green plasmids
- Send Mediprep DNA for sequencing
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- Screen Lyn and Syk-eYFP colonies
- Miniprep/digest verify CD79B-TCS-G4 pEXPR
- Grow up Gmab Heavy miniprep cultures from pENTR plate for sequencing
- LRs: CD79B-TCS-G4 (1), Gmab Light (1), Gmab Heavy (3, 4)
- Grow up midiprep cultures for CD79A, CD79A-TCS-G4, Syk+stop, Syk-TEVp
- Design AarI Primers
- Design 2 Lyn gBlock (1w/ stop and QX 1W/ fusion)
- Design new gBlock Primers
- Screen CD79B
- Organize Geneious
- How we chose linker links on Wiki
- Update Experiments list and expand on them
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- Design Digests (EE)
- Transform
- Pick Colonies
- More Sequencing
- Lyn gBlock (CR)
- Redesign Lyn fusions to Q sites (AS)
- Alternative Cloning (KRB)
- Organize Geneious
- How we chose linker links on Wiki
- Update Experiments list and expand on them
- Order DSM Oligos
- Are antibodies ordered?
- Pick colonies
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- How we chose linker links on Wiki (CR)
- Update experiments list and expand on them (CR, KRB)
- Update various parts pages on wiki with relevant digests (KRB)
- Transform LRs
- Lots of sequencing analysis (AS)
- LRs based on sequencing data
- Order Lyn Gblock W/ Nelson's PCR Flankers (CR)
- Order SDM Oligos for Lyn (EE)
- Organize lab boxes (CR)
- Organize Geneious (CR)
- MAKE SURE ANTIBODIES ARE ORDERED (KRB, CR)
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- anti Syk
- secondary western Ig
- anti IgM
- Design Sequencing Primers (CR)
- Restriction Digests for Orange Plasmids (EE)
- Gel for 7 Plasmids (EE) (AS)
- PCR CD79B's & Gmab (AS)
- Find all blue plasmids and put in BCR Box (CR)
- Golden Gates
- LRs
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- Miniprep GG Colonies
- Transform & plate LR plasmids
- Transform & plate GG plasmids
- Email Brian about Pfizer stuff
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Link To Research Page
Parts List
Links To EVERYTHING!