This year, our main experiments were conducted in vitro. The only species we use in our experiments is E. coli DH-5 alpha1, which is the chassis in our project. The genes we used contain two main parts, toehold switches and the genes of expressed proteins, which were both synthesized directly from biotech companies. We designed toehold switches ourselves based on three papers2,3,4. They can combine with different miRNA and then open their stem-loops. We chose GFP and invertase as our expression proteins. Both of toehold switches and expression proteins are not harmful to human beings and creatures. Our project would be completely safe under the situation of operating correctly.
Our final product is a detector of oral cancer. We design our kit, miRNA.doc, based on glucometer. The new kit can work safely as the original one who has passed the standards of medical equipment. One of our primary concern is the entry-level for using miRNA.doc. We attempted to simplify the process as much as we could. However, the use of chemicals is unavoidable and would require basic chemical safety knowledge to use the kit.
- Bryant FR. Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity. J Biol Chem. 1988 Jun 25;263(18):8716-23. PMID: 2967815.
- Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo-designed regulators of gene expression. Cell, 159(4), 925-939.
- Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., ... & Daringer, N. M. (2016). Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell, 165(5), 1255-1266.
- Wang, S., Emery, N. J., & Liu, A. P. (2019). A novel synthetic toehold switch for microRNA detection in mammalian cells. ACS synthetic biology, 8(5), 1079-1088.