Contribution

Table of contents

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

  1. CD19 BBa_K5062003
  2. FcyR BBa_K5062004
  3. MegF10 Costim - BBa_K5062005
  4. CD19/ FcyR Tandem - BBa_K5062006
  5. 5’ Hammerhead Ribozyme - BBa_K5062009
  6. 3’ Hepatitis D Virus Ribozyme - BBa_K5062010
  7. SIRPa shRNA - BBa_K5062011
  8. PD1 shRNA - BBa_K5062012
  9. SIRPa flank sequence - BBa_K5062013
  10. PD1 flank sequence - BBa_K5062014
  11. SIRPa Ribo-shRNA - BBa_K5062016
  12. PD-1 Ribo-shRNA - BBa_K5062017
  13. SIRPa/PD1 Multiplex shRNA - BBa_K5062018