Team:NMU China/Parts
Basic Parts
We designed 22 basic parts synthesized by GENEWIZ this year. We used these basic parts to synthesize composite parts.
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Favourite Parts
Our favorite basic part is BBa_K4040003 and our favorite composite part is BBa_K4040018.
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Improvement of existing parts
We made some improvements based on BBa_K3132026 from SMMU_China-2019 to reduce the background expression activity of the receptor.
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Basic Parts
We designed 17 basic parts in total.You can click on the part number to see the details of basic parts.
| Name | Type | Description |
|---|---|---|
| BBa_K4040000 | Protein_Domain | Intracellular Domain of the MEGF10 Protein |
| BBa_K4040001 | Protein_Domain | Intracellular Domain of the MERTK Protein |
| BBa_K4040002 | Protein_Domain | FcRγ |
| BBa_K4040003 | Protein_Domain | CD3 zeta chain |
| BBa_K4040004 | Protein_Domain | CD8 hinge |
| BBa_K4040005 | Tag | Myc tag |
| BBa_K4040006 | Protein_Domain | TEV protease |
| BBa_K4040007 | Protein_Domain | CD8TM |
| BBa_K4040011 | Protein_Domain | tTA |
| BBa_K4040013 | Protein_Domain | mIL-6R |
| BBa_K4040014 | Protein_Domain | CD8a signal peptide |
| BBa_K4040019 | Protein_Domain | CR3022 scFv |
| BBa_K4040032 | Protein_Domain | IL-6 signal transducer, gp130 |
| BBa_K4040034 | Protein_Domain | Truncated HER2 |
| BBa_K4040035 | Protein_Domain | Truncated EGFR |
| BBa_K4040036 | Protein_Domain | CD20 |
| BBa_K4040037 | Protein_Domain | T2A peptide |
Composite Parts
We designed 10 composite parts in total to constitute our project. You can click on the part number to see the details of composite parts.
| Name | Type | Description |
|---|---|---|
| BBa_K4040015 | Composite | CAR-MEGF10 |
| BBa_K4040016 | Composite | CARγ |
| BBa_K4040017 | Composite | CAR ζ |
| BBa_K4040018 | Composite | CAR-MERTK |
| BBa_K4040020 | Composite | GP130-TEV |
| BBa_K4040021 | Composite | IL6R-TCS-Gal4-KRAB |
| BBa_K4040022 | Composite | IL6R-TCS-tTA |
| BBa_K4040023 | Composite | pSV40-UAS-GFP Expression Cassette |
| BBa_K4040024 | Composite | pTET-mCherry-PGK-CD20 Expression Cassette |
| BBa_K4040033 | Composite | UAS-pSV40 |
Favourite Parts
Our favorite Basic Part is CD3 zeta chain (BBa_K4040003), which encodes for the CAR-CD3Z intracellular segment. This part successfully transmits CAR-perceived signals into the cell, triggering subsequent cascades that activate CAR-M phagocytosis and induce the release of inflammatory factors. CD3Z intracellular segment has been widely used in CAR-T intracellular segment, and intracellular segment for CAR-M has also been reported in previous literature. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways [1,2].
Future iGEM teams working with CAR can make use of it to create CAR-Ts and CAR-Ms which induce phagocytosis and immune response. As seen in our results, CARzeta macrophages phagocytosed Spike-bearing 293 cells in an S-specific manner. CARzeta mediated significantly stronger levels of SARS-CoV-2 phagocytosis by THP-1 cells than CARD（truncated CAR）.
Our favourite composite part is CARMERTK (BBa_K4040018), which was regarded as an unsuccessful receptor in a previous report [3] and showed no cellular killing effect on target cells when expressed in THP-1 cells in our assay, demonstrated a virion clearance capacity similar to that of CARγand CARzeta. Our data further support that CARMERTK mediates ‘immunologically silent' virion removal, which does not elicit a proinflammatory response. It opens the possibility for future iGEM teams to build such CAR without strong inflammatory response.
Improvement of existing parts
We made some improvements based on BBa_K3132016 from SMMU_China-2019 to reduce the background expression activity of the receptor. You can click on the part number to see the details of improvement.
| Name | Type | Description |
|---|---|---|
| BBa_K4040025 | Protein_Domain | GEMS |
| BBa_K4040026 | Protein_Domain | GEMS with one Alanine added |
| BBa_K4040027 | Protein_Domain | GEMS with two Alanines added |
| BBa_K4040028 | Protein_Domain | GEMS with three Alanines added |
| BBa_K4040029 | Protein_Domain | GEMS with four Alanines added |
References
[1]Barber EK, Dasgupta JD, Schlossman SF, Trevillyan JM, Rudd CE. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc Natl Acad Sci U S A. 1989 May;86(9):3277-81. doi: 10.1073/pnas.86.9.3277. PMID: 2470098; PMCID: PMC287114.
[2]Iwashima M, Irving BA, van Oers NS, Chan AC, Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994 Feb 25;263(5150):1136-9. doi: 10.1126/science.7509083. PMID: 7509083.
[3]MorrisseyMA,etal.ChimericAntigenReceptorsThatTriggerPhagocytosis.Elife(2018)7:e36688.doi:10.7554/eLife.36688