Parts

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:

BBa_K3878021 encoding for hsa-miRNA-141-5p
BBa_K3878022 encoding for hsa-miRNA-200c
BBa_K3878020encoding for hsa-miRNA-21-5p

Due to lack of time, we did not characterize these parts.

Team:UParis BME/Parts

Parts