Our project relies on microRNA detection using toehold switches. These de-novo-designed riboregulators are composed of several parts:
The first part we engineered is the BBa_K3878000, it is the plasmid encoding for the repressed gene of our toehold switch. We cloned GFP into pET24-d(+) plasmid using restriction enzymes XhoI and EcorI. We built this part with the idea of making it a host plasmid for all our toehold switch candidates. We can order the variable part of our modeled toehold switch and clone it into this part before the GFP.
Each of the variable parts of the previously designed toehold switches are considered as a part itself. We engineered these parts with T7 promoter leading to its RNA expression in bacteria with T7 RNA polymerase and restriction sites for molecular cloning into plasmid encoding repressor genes such as the parts BBa_K3878000 (cloning using restriction site) or BBa_K3453101 (cloning using golden gate assembly).
Table 1: These parts are DNA fragments that encode toehold switch candidates targeting hsa-miR-141-5p. They contain the BamH1 and BglII restriction sites.
| Name of the part |
Component |
| BBa_K3878001 |
Encodes the variable part for the Toehold Switch Candidates 1 |
| BBa_K3878008 |
Encodes the variable part for the Toehold Switch Candidates 2 |
| BBa_K3878009 |
Encodes the variable part for the Toehold Switch Candidates 3 |
| BBa_K3878010 |
Encodes the variable part for the Toehold Switch Candidates 4 |
| BBa_K3878011 |
Encodes the variable part for the Toehold Switch Candidates 5 |
| BBa_K3878012 |
Encodes the variable part for the Toehold Switch Candidates 6 |
Table 2: These parts are DNA fragments that encode toehold switch candidates targeting hsa-miR-21-5p. They contain homologous sequences including BsaI restriction sites to be assembled into a golden gate plasmid such as BBa_K3453101.
| Name of the part |
Component |
| BBa_K3878007 |
Encodes the variable part for the Toehold Switch Candidates 1 |
| BBa_K3878014 |
Encodes the variable part for the Toehold Switch Candidates 2 |
| BBa_K3878015 |
Encodes the variable part for the Toehold Switch Candidates 3 |
| BBa_K3878013 |
Encodes the variable part for the Toehold Switch Candidates 4 |
| BBa_K3878016 |
Encodes the variable part for the Toehold Switch Candidates 5 |
| BBa_K3878017 |
Encodes the variable part for the Toehold Switch Candidates 6 |
| BBa_K3878018 |
Encodes the variable part for the Toehold Switch Candidates 7 |
To test our toehold switch, we engineered parts encoding for target human miRNAs. These parts encodes for the miRNAs via T7 RNA polymerase:
Due to lack of time, we did not characterize these parts.
