Our Contribution to Future Teams
Although the experimental validation of our pTRE3G-dCas9-VP64-3xFLAG (NTD) construct has not yet demonstrated functional expression in mammalian cells, the development process itself represents a meaningful contribution to the iGEM registry and future synthetic biology work. The plasmid design, cloning workflow, and bacterial propagation protocols collectively establish a reproducible framework for inducible CRISPRa systems that can be adapted or improved upon by future teams.
Vector Propagation and Stability
Our vector was successfully propagated in E. coli DH5α, exhibiting high copy number yields under standard antibiotic selection conditions. Across multiple preparations, we achieved an average plasmid concentration of approximately 400 ng/μL, validating both the stability and recoverability of the construct. Furthermore, our refined transformation and culture parameters—specifically the use of 1:1 to 1:2 serial dilutions and a 12-hour incubation period—produced consistently well-isolated colonies, offering a practical optimization for handling large plasmids in DH5α. These results reinforce that the vector backbone is structurally intact and can be efficiently cloned, stored, and distributed for further research.
Valuable Insights from Expression Challenges
Although preliminary BCA assays and Western blot data revealed no conclusive dCas9-VP64 expression in A549 cells, this negative result provides valuable insight into potential challenges in expressing large, multi-domain proteins under inducible promoters such as TRE3G. Our data underscore the importance of optimizing Doxycycline dosage, promoter strength, and plasmid ratios in Tet-ON systems, factors that future teams can investigate to achieve more reliable induction.
A Foundation for Future Work
In combination with our sgRNA library, the pTRE3G-dCas9-VP64-3xFLAG (NTD) construct establishes a foundation for a modular CRISPRa activation system designed for precise and tunable control of antiviral or immunomodulatory genes. Future refinement of our system allows its quick adaptation to any set of genes, simply by changing the sgRNAs, allowing for the potential to develop a quickly inducible, broad-spectrum, and transient gene activation method, not just for immunomodulation.
Even though expression was not observed under our tested conditions, the documentation, characterization workflow, and troubleshooting steps outlined in this project contribute essential procedural knowledge to the iGEM community. By sharing both our successes in plasmid preparation and our limitations in expression, we hope to enable others to refine, adapt, and expand upon our system toward effective inducible gene activation in mammalian models.