Overview
In the effort to produce an effective solution for hard-to-treat cancers, CAR-Ma was developed as a multiplexed and modular system. Due to the advanced and complex nature of CAR_Ma, an undergraduate team developing this over summer would be an insurmountable task. CAR_Ma was built from the ground-up to have design and engineering teams leapfrogging to ensure constant development and building.
Plasmid Designs, Assembly and Parts
CAR-Ma relies heavily on the composition of tested and novel biotechnologies. All the
components we used and developed were referenced to existing parts on the registry or were
given new parts on their own. A pool of referenced literature of whom which we called upon
will be provided, due to the complexity of this project, the referenced literature will
touch upon topics related to the design of our CAR complex, our nanobody sequences,
optimized human co-stimulatory domain and our gene modulation pathways. We also provide
cloning information in order to allow future members to design multi-modal RNA for their own
purposes using some or all of our parts.
Aside from the deluge of literature compiled in relevance to this topic we have several
other components which contribute to future attempts at CAR_Ma. Our full plasmid designs
will be made available: This includes primer sequences, recommended Tm values and PCR
protocols used throughout our project. Cloning of the control system into srRNA is only
feasible done through NEBuilder HiFI DNA Assembly due to the high demands of the system. Our
compiled primers also include the troubleshooting information as well as specific run
protocols due to the irregular nature of our primers. A simplified cloning scheme is also
included for the individual insertion of the control section or 2x K-Turn into either
plasmid.
Download our Snapgene files here!
In regards to the development of parts that we contribute. We are contributing shRNA
sequences and pairing them with ribozymes for their effective release. We are also including
our own human co-stimulatory domains inspired by Morrissey et al. (2018) for uses in human
macrophages and therapeutics, since the original paper utilizes mouse co-stimulatory domains
for mouse macrophages.
Novel Part Contributions
- CD19 BBa_K5062003
- FcyR BBa_K5062004
- MegF10 Costim - BBa_K5062005
- CD19/ FcyR Tandem - BBa_K5062006
- 5’ Hammerhead Ribozyme - BBa_K5062009
- 3’ Hepatitis D Virus Ribozyme - BBa_K5062010
- SIRPa shRNA - BBa_K5062011
- PD1 shRNA - BBa_K5062012
- SIRPa flank sequence - BBa_K5062013
- PD1 flank sequence - BBa_K5062014
- SIRPa Ribo-shRNA - BBa_K5062016
- PD-1 Ribo-shRNA - BBa_K5062017
- SIRPa/PD1 Multiplex shRNA - BBa_K5062018